Cephalosporin C acylase

ABSTRACT

PCT No. PCT/JP94/01799 Sec. 371 Date May 1, 1996 Sec. 102(e) Date May 1, 1996 PCT Filed Oct. 26, 1994 PCT Pub. No. WO95/12680 PCT Pub. Date May 11, 1995A mutant CC acylase wherein at least one amino acid at the Ala49, Met164, Ser166, Met174, Glu358, Met465, Met506, or Met750 position of the amino acid sequence of the native CC acylase is replaced by a different amino acid, a DNA coding therefor, an expression vector containing the said DNA, a microorganism transformed with the said expression vector, the production of the CC acylase by culturing the said transformant, and use thereof for the production of a compound. The mutant CC acylase of the invention has desirable properties in terms of enzymatic potency, alteration of pH profile, efficiency of processing, and the like.

The present application is a national stage application based onPCT/JP94/01799, filed Oct. 26, 1994.

The invention relates to a new cephalosporin C acylase (hereinafterreferred to as "CC acylase"). More particularly, it relates to a newmutant CC acylase produced by protein engineering, a DNA codingtherefor, an expression vector containing the said DNA, a microorganismtransformed with the said expression vector, and the production of theCC acylase by culturing the said transformant.

The cephalosporin C acylase is a general term for an enzyme, which is,in common, capable of hydrolyzing cephalosporin C to7-aminocephalosporanic acid (7-ACA).

Hitherto, there have been found three enzymes which should be classifiedas CC acylase, namely Cephalosporin C acylases SE83, N176 and V22, aminoacid sequences of which are disclosed in Journal of Fermentation andBioengineering Vol. 72, 232-243 (1991). In this literature, numbering ofthe amino acid sequence of CC acylase is begun at the methionine groupof the N-terminal portion thereof. However, numbering of the amino acidsequence of CC acylase is begun at the threonine group adjacent to themethionine group of the N-terminal portion thereof in thisSpecification, because the N-terminal methionine of α-subunit of matureCC acylase obtained by expressing CC acylase gene in prokaryote isremoved by an enzyme (e.g. aminopeptidase) to give a mature CC acylasehaving the threonine group as the N-terminal amino acid thereof.Production of native type CC acylase by recombinant DNA technology isalso disclosed in the said literature and it has been found that theexpressed CC acylase is intracellularly processed to give an active formcomposed of α-subunit and β-subunit. However, efficiency of theprocessing is generally low in E. coli. From the results of extensivestudies, the inventors of this invention have succeeded in producingmutant CC acylases which have more desirable properties which arecharacterized by higher enzymatic potency, alteration of pH profile,higher efficiency of processing and the like.

The new mutant CC acylase of this invention can be characterized by thefollowing.

A mutant CC acylase wherein at least one amino acid at the Ala⁴⁹,Met¹⁶⁴, Ser¹⁶⁶, Met¹⁷⁴, Glu³⁵⁸, Met⁴⁶⁵, Met⁵⁰⁸ or Met⁷⁵⁰ position of theamino acid sequence of the native CC acylase is replaced by a differentamino acid.

Preferred examples of the different amino acid to replace Met¹⁶⁴ mayinclude neutral amino acids such as glycine, alanine, leucine and thelike.

Preferred examples of the different amino acid to replace Ser¹⁶⁶,Met¹⁷⁴, Met⁴⁶⁵, Met⁵⁰⁶ and/or Met⁷⁵⁰ may include neutral amino acidssuch as alanine and the like.

Preferred examples of the different amino acid to replace Glu³⁵⁸ mayinclude neutral-amino acids (e.g. isoleucine, etc.), basic amino acids(e.g. lysine, etc.) and the like.

Most preferable example of the different amino acid to replace Ala⁴⁹ isleucine.

The mutant CC acylase of this invention may also be a mutant CC acylaseprepared by replacing at least one amino acid at another position of theamino acid sequence of native CC acylase with a different amino acid,for example, by replacing Met²⁶⁹ and/or Cys³⁰⁵ of the mutant CC acylasewith (a) different amino acid(s).

A preferred example of the mutant CC acylase can be represented by thefollowing formula in its precursor form before processing into α-subunitand β-subunit thereof:

A1-48-X1-A50-163-X2-Gly-X3-A167-173-X4-A175-357-X5-A359-464-X6-A466-505-X7-A507-749-X8-A751-773

wherein A1-48 is the same amino acid sequence as that from Thr¹ to Glu⁴⁸of native CC acylase,

A50-163 is the same amino acid sequence as that from Asp⁵⁰ to Leu¹⁶³ ofnative CC acylase,

A167-173 is the same amino acid sequence as that from Val¹⁶⁷ to Arg¹⁷³of native CC acylase,

A175-357 is the same amino acid sequence as that from Leu¹⁷⁵ to Val³⁵⁷of native CC acylase,

A359-464 is the same amino acid sequence as that from Thr³⁵⁹ to Ala⁴⁶⁴of native CC acylase,

A466-505 is the same amino acid sequence as that from Pro⁴⁶⁶ to Ile⁵⁰⁵of native CC acylase,

A507-749 is the same amino acid sequence as that from Lys⁵⁰⁷ to Ala⁷⁴⁹of native CC acylase,

A751-773 is the same amino acid sequence as that from Val⁷⁵¹ to Ala⁷⁷³of native CC acylase,

X1 is Ala or a different amino acid,

X2, X4, X6, X7 and X8 are each Met or a different amino acid,

X3 is Ser or a different amino acid and

X5 is Glu or a different amino acid,

providing that Met²⁶⁹ and/or Cys³⁰⁵ may be replaced by (a) differentamino acid(s), and when X1 is Ala, X2, X4, X6, X7 and X8 are each Met,X3 is Ser and X5 is an amino acid other than Glu.

In this specification, a nomenclature for naming a specific mutant CCacylase is conveniently employed. According to this nomenclature, forexample, a mutant CC acylase which is prepared by replacing themethionine residue at position 164 of the amino acid sequence of nativeCC acylase with leucine should be designated as a mutant CC acylaseM164L, in which M is a one-letter abbreviation of the methionine (anamino acid) residue to be replaced, 164 is a position number of theamino acid sequence of native CC acylase and L is a one-letterabbreviation of leucine (the different amino acid) used for replacingthe methionine (the former amino acid) residue. On the other hand, forexample, mutant CC acylases. M164L and M164A are prepared by replacingthe methionine residue at position 164 of the amino acid sequence ofnative CC acylase with leucine and alanine, respectively. A mutant CCacylase M164L/M174A/M269Y is prepared by replacing the methionineresidue at position 164 of the amino acid sequence of native CC acylasewith leucine, the methionine residue at position 174 of the amino acidsequence of native CC acylase with alanine and the methionine residue atposition 269 of the amino acid sequence of native CC acylase withtyrosine.

The mutant CC acylase of this invention can be prepared by recombinantDNA technology, polypeptide synthesis and the like.

Namely, the new CC acylase can be prepared by culturing a host celltransformed with an expression vector comprising DNA encoding amino acidsequence of the new CC acylase in a nutrient medium and recovering thenew CC acylase from the cultured broth.

Particulars of this process are explained in more detail as follows.

The host cell may include microorganisms bacteria (e.g. Escherichiacoli, Bacillus subtilis, etc.), yeast (e.g. Saccharomyces cerevisiae,etc.), animal cell lines and cultured plant cells!. Preferred examplesof the microorganism may include bacteria, especially a strain belongingto the genus Escherichia (e.g. E. coli JM109 ATCC 53323, E. coli HB101ATCC 33694, E. coli HB101-16 FERM BP-1872, E. coli 294 ATCC 31446,etc.), yeast, especially a strain belonging to the genus Saccharomycese.g. Saccharomyces cerevisiae AH22!, animal cell lines e.g. mouseL929-cell, Chinese hamster ovary (CHO) cell, etc.! and the like.

When a becterium, especially E. coli is used as a host cell, theexpression vector is usually composed of at least promoter-operatorregion, initiation codon, DNA encoding amino acid sequence of the new CCacylase, termination codon, terminator region and replicatable unit.When yeasts or animal cells are used as host cells, the expressionvector is preferably composed of at least promoter, initiation codon,DNA encoding amino acid sequences of the signal peptide and the new CCacylase, and termination codon. It is possible that enhancer sequence,5'- and 3'-noncoding region of the new CC acylase, splicing junctions,polyadenylation site and replicatable unit are also inserted into theexpression vector.

The promoter-operator region comprises promoter, operator andShine-Dalgarno (SD) sequence (e.g. AAGG, etc.). Preferablepromoter-operator region may include conventionally employedpromoter-operator region (e.g. PL-promoter and trp-promoter for E. coli)and promoter of the CC acylase N-176 chromosomal gene. The promoter forexpression of the new CC acylase in yeast may include the promoter ofthe TRP1 gene, the ADHI or ADHII gene and acid phosphatase (pH05) genefor S. cerevisiae and the promoter for the expression of the new CCacylase in mammalian cells may include SV40 early or late-promoter,HTLV-LTR-promoter, mouse metallothionein I(MMT)-promoter,vaccinia-promoter and the like.

Preferable initiation codon may include methionine codon (ATG).

The signal peptide may include a signal peptide of conventionallyemployed other enzymes (signal peptide of the native t-PA, signalpeptide of the native plasminogen) and the like.

The DNA encoding amino acid sequence of the new CC acylase can beprepared in a conventional manner such as a partial or whole DNAsynthesis using DNA synthesizer and/or treatment of the complete DNAsequence coding for the native CC acylase inserted in a suitable vector(e.g. pCCN 176-2) obtainable from a transformant e.g. E. coli JM109(pCCN 176-2) FERM BP-3047! in a suitable manner such as a conventionalmutation method e.g. cassette mutation method (cf. Tokunaga, T. et al.,Eur. J. Biochem. 153, 445-449 (1985)), PCR mutation method (cf. Higuchi,R. et al., Nucleic Acids Res. 16, 7351-7367 (1988)), Kunkel's method(cf. Kunkel, T. A. et al., Methods Enzymol. 154, 367 (1987)) and thelike! in addition to treatment with a suitable enzyme (e.g. restrictionenzyme, alkaline phosphatase, polynucleotide kinase, DNA ligase, DNApolymerase, etc.).

The termination codon(s) may include a conventionally employedtermination codon (e.g. TAG, TGA, etc.).

The terminator region may include natural or synthetic terminator (e.g.synthetic fd phage terminator, etc.).

The replicatable unit is a DNA compound capable of replicating the wholeDNA sequence belonging thereto in a host cell, and may include naturalplasmid, artificially modified plasmid (e.g. DNA fragment prepared fromnatural plasmid) and synthetic plasmid. Preferable examples of theplasmid may include plasmid pBR322 and the artificially modified thereof(DNA fragment obtained from a suitable restriction enzyme treatment ofpBR322) for E. coli, yeast 2μ plasmid and yeast chromosomal DNA foryeast, plasmid pRSVneo ATCC 37198, plasmid pSV2dhfr ATCC 37145, plasmidpdBPV-MMTneo ATCC 37224, and plasmid pSV2neo ATCC 37149 for mammaliancells.

The enhancer sequence may include the enhancer sequence (72 b.p.) ofSV40.

The polyadenylation site may include the polyadenylation site of SV40.

The splicing junction may include the splicing junction of SV40.

The promoter, initiation codon, DNA encoding amino acid sequence of thenew CC acylase, termination codon(s) and terminator region canconsecutively and circularly be linked together with an adequatereplicatable unit (plasmid), if desired, using (an) adequate DNAfragment(s) (e.g. linker, other restriction site, etc.) in aconventional manner (e.g. digestion with restriction enzyme, ligationusing T4 DNA ligase) to give an expression vector.

A host cell can be transformed (transfected) with the expression vector.Transformation (transfection) can be carried out in a conventionalmanner e.g. Kushner method for E. coli, calcium phosphate method formammalian cells, microinjection, etc.! to give a transformant(transfectant).

For the production of the new CC acylase by the process of thisinvention, the thus obtained transformant comprising the expressionvector is cultured in an aqueous nutrient medium.

The nutrient medium may contain carbon source(s) (e.g. glucose,glycerine, mannitol, fructose, lactose, etc.) and inorganic or organicnitrogen source(s) (e.g. ammonium sulfate, ammonium chloride,hydrolysate of casein, yeast extract, polypeptone, bactotrypton, beefextract, etc.). If desired, other nutritious sources e.g. inorganicsalts (e.g. sodium or potassium biphosphate, dipotassium hydrogenphosphate, magnesium chloride, magnesium sulfate, calcium chloride),vitamins (e.g. vitamin B1), antibiotics (e.g. ampicillin, kanamycin),etc.! may be added to the medium. For the culture of mammalian cells,Dulbecco's Modified Eagle's Minimum Essential Medium (DMEM) supplementedwith fetal calf serum and an antibiotic is often used.

The culture of the transformant (including transfectant) is usuallycarried out at pH 5.5-8.5 (preferably pH 7-7.5) and 18°-40° C.(preferably 20°-30° C.) for 5-50 hours.

When the thus produced new CC acylase exists in the culture solution,culture filtrate (supernatant) is obtained by filtration orcentrifugation of the cultured broth. From the culture filtrate, the newCC acylase can be purified in a conventional manner generally employedfor the purification and isolation of natural or synthetic proteins(e.g. dialysis, gel filtration, affinity column chromatography usinganti-CC acylase monoclonal antibody, column chromatography on a suitableadsorbent, high performance liquid chromatography, etc.). When theproduced new CC acylase exists in periplasm and cytoplasm of thecultured transformant, the cells are collected by filtration andcentrifugation, and the cell wall and/or cell membrane thereof are(is)destroyed by, for example, treatment with super sonic waves and/orlysozyme to give debris and/or lysate. The debris and/or lysate can bedissolved in a suitable aqueous solution (e.g. 8M aqueous urea, 6Maqueous quanidium salts). From the solution, the new CC acylase can bepurified in a conventional manner as exemplified above.

This invention further provides a process for the preparation of acompound of the formula: ##STR1## wherein R¹ is acetoxy its salt, orhydrogen, or its salt, which comprises contacting a compound of theformula: ##STR2## wherein R¹ is as defined above and R² is carboxylicacyl, or its salt,

with the cultured broth of a microorganism transformed with anexpression vector comprising DNA encoding the new CC acylase of thisinvention or its processed material.

The carboxylic acyl for R² may include aliphatic, aromatic orheterocyclic carboxylic acyl and suitable example thereof may be C₁ -C₆alkanoyl which may have one or two suitable substituent(s) selected fromthe group of amino, hydroxy, carboxy, C₁ -C₆ alkanoylamino, benzamido,thienyl, and the like.

Suitable salt of the compounds (I) and (II) may be alkali metal salt(e.g. sodium salt, potassium salt, lithium salt).

If the CC acylase activity usually exists in transformed cells, thefollowing preparations can be exemplified as a processed material of thecultured broth.

(1) Raw cells: separated from the cultured broth in a conventionalmanner such as filtration and centrifugation;

(2) dried cells: obtained by drying said raw cells in a conventionalmanner such as lyophilization and vacuum drying;

(3) cell-free extract: obtained by destroying said raw or dried cells ina conventional manner (e.g. autolysis of the cells using an organicsolvent, grinding the cells with alumina, sea sand, etc. or treating thecells with super sonic waves);

(4) enzyme solution: obtained by purification or partial purification ofsaid cell-free extracts in a conventional manner (e.g. columnchromatography); and

(5) immobilized cells or enzyme: prepared by immobilizing said cells orenzyme in a conventional manner (e.g. a method using acrylamide, glassbead, ion exchange resin, etc.).

The reaction comprising a contact of the compound (II) with the enzymecan be conducted in an aqueous medium such as water or a buffersolution, that is, it can be usually conducted by dissolving orsuspending the cultured broth or its processed material in an aqueousmedium such as water or a buffer solution containing the compound (II).

Preferable pH of the reaction mixture, concentration of the compound(II), reaction time and reaction temperature may vary with properties ofthe cultured broth or its processed material to be used. Generally, thereaction is carried out at pH 6 to 10, preferably pH 7 to 9, at 5° to40° C., preferably 5° to 37° C. for 0.5 to 50 hours.

The concentration of the compound (II) as a substrate in the reactionmixture may be preferably selected from the range of from 1 to 100mg/ml.

The thus produced compound (I) can be purified and isolated from thereaction mixture in a conventional manner.

Specific activities of mutant acylases were determined according to theprocedure mentioned below.

i) GL-7ACA acylase activity

To 500 μl of GL-7ACA solution 10 mg/ml in 0.15M Tris.HCl (pH 7.5)! thatwas pre-incubated at 37° C. for 10 min, 20 μl of sample acylase wasadded and the mixture was incubated at 37° C. for 5 min. The reactionwas stopped by the addition of 550 μl of 5% acetic acid. Aftercentrifugation (10,000 rpm for 5 min at ambient temperature) of theresulting mixture, the supernatant was used for the assay of 7ACAformation.

HPLC conditions: column: TSKgel ODS-80 TMCTR 4.4 mm×100 mm (TOSOH);eluate: 100 mM citric acid, 5.0 mM sodium n-hexane-1-sulfonate in 14.3%(V/V) acetonitrile; flow rate: 1.0 ml/min; injection volume: 10 μl;detector: 254 nm.

One unit was defined as the activity capable of synthesizing 1.0 μmoleof 7ACA from GL-7ACA per minute at 37° C.

ii) CC acylase activity

To 500 μl of CC solution 10 mg/ml sodium salt of cephalosporin C in0.15M Tris.HCl (pH 8.7), pH was readjusted with 1N NaOH to pH 8.7! thatwas pre-incubated at 37° C. for 10 min, 20 μl of a sample acylase wasadded and the mixture was incubated at 37° C. for 10 min. The reactionwas stopped by the addition of 550 μl of 5% acetic acid. Aftercentrifugation (10,000 rpm for 5 min at ambient temperature) of theresulting mixture, the supernatant was used for the assay of 7ACAformation.

HPLC conditions: column: TSKgel ODS-80 TMCTR 4.4 mm×100 mm (TOSOH),eluate: 100 mM citric acid, 5.0 mM sodium n-hexane-1-sulfonate in 14.3%(V/V) acetonitrile; flow rate: 1.0 ml/min;

injection volume: 20 μl; detector: 254 nm.

One unit was defined as the activity capable of synthesizing 1.0 μmoleof 7ACA from sodium salt of Cephalosporin C per minute at 37° C.

Brief explanation of the accompanying drawings is as follows.

FIG. 1 shows DNA oligomers used in the working Examples of thisSpecification (SEQ ID NO:1-42).

FIG. 2 is a schematic presentation of the construction of pCC001A.

FIG. 3 is a schematic presentation of the construction of pCC002A.

FIG. 4 is a schematic presentation of the construction of pCK002.

FIG. 5 is a schematic presentation of the construction of pCC007A andpCCNt013.

FIG. 6 is a schematic presentation of the construction of pCC013A.

FIG. 7 is a schematic presentation of the construction of pΔN176 andpCK013.

FIG. 8 is a schematic presentation of the preparation of mp18p181 andmp18p183.

FIG. 9 is a schematic presentation of the preparation of mp18p181M164A,mp18p181M174A, pCKM174A and pCKM164A.

FIG. 10 is a schematic presentation of the preparation of pCKS166A.

FIG. 11 is a schematic presentation of the preparation of pCKM164L andpCKM164G.

FIG. 12 is a schematic presentation of the construction of mp19pfu62.

FIG. 13 is a schematic presentation of the preparation of RF DNAs(mp19pfu62M465A, mp19pfu62M506A and mp19pfu62M750A) and expressionvectors (pCKM465A, pCKM506A and pCKM750A).

FIG. 14 is a schematic presentation of the preparation of p269I358K,p269I358S and p269I358L.

FIG. 15 is a schematic presentation of the preparation of pCCE358R andpGCE358T.

FIG. 16 is a schematic presentation of the preparation of p164L269Y,p164L269F and p164L269Y305S.

FIG. 17 is a schematic presentation of the preparation of p164L174A andp164A174A.

FIG. 18 is a schematic presentation of the preparation of p164A269Y,p164L174A269Y, p164L174A269F, p164A174A269Y305S.

FIG. 19 is a schematic presentation of the preparation ofp164L174A269Y305S750A.

FIG. 20 is a schematic presentation of the preparation of pCKA49L.

FIG. 21 is a schematic presentation of the preparation ofp49L164L174A269Y.

FIG. 22 shows the nucleotide and amino acid sequences of mutant CCacylase M164A (SEQ ID NO:43,44).

FIG. 23 shows the nucleotide and amino acid sequences of mutant CCacylase S166A (SEQ ID NO:45,46).

FIG. 24 shows the nucleotide and amino acid sequences of mutant CCacylase M269I/E358K (SEQ ID NO:47,48).

FIG. 25 shows the nucleotide and amino acid sequences of mutant CCacylase M164L/M174A/M269Y (SEQ ID NO:49,50).

FIG. 26 shows the nucleotide and amino acid sequences of mutant CCacylase A49L (SEQ ID NO:51,52).

In the following Examples, some plasmids, enzymes, such as restrictionenzymes, T4 DNA ligases, and other materials were obtained fromcommercial sources and used according to the indication by suppliers. Inparticular, plasmid pCCN176-2 and plasmid pTQiPAΔtrp used as startingmaterials in Examples have been deposited at an internationaldepository, National Institute of Bioscience and Human-Technology,Agency of Industrial Science and Technology in Japan, as transformant E.coli JM109 (pCCN176-2) FERM BP-3047 and transformant E. coli HB101-16(pTQiPAΔtrp) FERM BP-1870, respectively, and can be easily obtainedbased on U.S. Pat. No. 5,192,678 and European Patent ApplicationPublication No. 302456. Other plasmid etc. can be easily preparedaccording to the description in the specification, from said pCCN176-2,pTQiPAΔtrp and those commercially available. Operations employed for thecloning of DNA, transformation of host cells, cultivation oftransformants, recovery of the new CC acylase from the cultured broth,and the like are well known in the art or can be adapted fromliteratures.

The following examples are given for the purpose of illustrating thisinvention, and the invention is not limited thereto.

EXAMPLE 1 (Synthesis of Oligodeoxyribonucleotide)

A DNA oligomer SO-M164A as listed in FIG. 1(a)! was synthesized with381A DNA synthesizer (Applied Biosystems Inc.). The DNA was liberatedfrom CPG polymer support (CPG: controlled pore glass) with 28% aqueousammonia followed by heating at 60° C. for 9 hours to remove allprotection groups (SEQ ID NO:2). The reaction mixture was evaporated invacuo, and the residue was dissolved in 200 μl of TE buffer 10 mMTris.HCl (pH 7.4)-1 mM EDTA!. The resulting crude DNA solution wasapplied to reverse phase HPLC column; COSMOSIL C18 4.6 mm×150 mm(Nacalai Tesque), eluate; A: 0.1M Triethylammonium acetate buffer (pH7.2-7.4), B: acetonitrile, gradient; initial A(100%), finalA(60%)+B(40%), linear gradient during 25 min, flow rate; 1.2 ml/min!.The eluate containing the objective DNA oligomer was collected andevaporated in vacuo. The purified DNA was dissolved in 200 μl of TEbuffer and stored at -20° C. before use.

All other DNA oligomers listed in FIG. 1 were synthesized and purifiedin a manner similar to that described above.

EXAMPLE 2 (Preparation of Expression Vector for Native CC Acylase N176Under the Control of trp Promoter)

(1) Construction of pCC002A, an ampicillin resistant expression vectorfor native CC acylase N176

(i) Construction of pCC001A

Plasmid pCCN176-3 preparation method of this plasmid from plasmidpCCN176-2 (which is obtainable from a transformant Escherichia coliJM109 (pCCN176-2) FERM BP-3047 in a conventional manner) is disclosed inpage 235 of JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 72, 1991!(1.0 μg) was digested with EcoRI and HindIII, and the 2.9 kb fragmentcarrying the entire coding region of CC acylase N176 was isolated byagarose gel electrophoresis. On the other hand, PTQiPAΔtrp (1.0 μg), anexpression vector for a mutant t-PA which is obtainable from atransformant, Escherichia coli HB101-16 (pTQiPAΔtrp) FERM BP-1870 in aconventional manner and a preparation method of which is disclosed inEuropean Patent Application Publication No. 3024561! was digested withXmaI and XhoI, and a larger DNA (5113 bp) was isolated. Synthetic DNAoligomers CT-1(5'-CCGGGTGTGTACACCAAGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCGACCGTGA),CT-2(5'-AGCTTCACGGTCGCATGTTGTCACGAATCCAGTCTAGGTAGTTGGTAACCTTGGTGTACACAC),TR-1 (5'-AGCTTGTCCTCGAGATCAATTAAAGGCTCCTTTTGGAGCCTTTTTTTTTTG) and TR-2(5'-TCGACAAAAAAAAAAGGCTCCAAAAGGAGCCTTTAATTGATCTCGAGGACA) werephosphorylated with T4 polynucleotide kinase and ligated with T4 DNAligase to give XmaI/SalI DNA fragment (114 bp) (SEQ ID NO:53-56). Theresultant DNA fragment was ligated to the XmaI/XhoI DNA to givepTQiPAdtrp. The pTQiPAdtrp (1.0 μg) was digested with EcoRI and HindIII.The resulting 4.3 kb DNA carrying trp promoter, a part of t-PA codingregion (Cys92 to Trp113) and the duplicated sequence of fd phage centralterminator were isolated. The 2.9 kb and 4.3 kb DNA fragments were mixedto ligate in the presence of T4 DNA ligase (300 units, Takara Shuzo) at16° C. for 5 hours. in 40 μl of a ligation buffer consisting of 50 mMTris.HCl 10 mM MgCl₂, 10 mM dithiothreitol and 1 mM ATP. The ligationmixture was used to transform E. coli JM109. The desired plasmid,designated as pCC001A, was obtained from one of the transformantsresistant to ampicillin and characterized by restriction mapping.

(ii) Construction of pCC002A

Plasmid pCC001A contains a portion of t-PA (Cys92 to Trp113) genebetween trp promoter and the acylase gene. In order to remove thisregion, pCC001A (1.0 μg) was digested with ClaI and MluI and theresulting 6.1 kb DNA fragment was isolated. On the other hand, pCCN176-3(1 μg) was digested with MluI and Sau3AI to isolate 189 bp DNA codingfor Asp7 to Arg71 of the acylase. Synthetic DNA oligomers 002a and 002b(0.5 nmole, respectively, Table 1 below) were phosphorylated with T4polynucleotide kinase (1.5 units, Takara Shuzo) in 10 μl of a buffer(kination buffer; 50 mM Tris.HCl, 10 mM MgCl₂, 10 mM DTT, 1.0 mM ATP) at37° C. for 1 hour and the reaction mixture was heated at 55° C. for 20min to inactivate the enzyme (SEQ ID NO:60,61). The resulting mixturewas combined to ligate with the 189 bp Sau3AI/MluI DNA in the presenceof T4 ligase at 15° C. for 3 hours in 20 μl of a ligation buffer. To theresultant ligation mixture, the 6.1 kb ClaI/MluI DNA fragment was addedand the mixture was incubated at 4° C. for 16 hours in the presence ofadditional T4 DNA ligase (300 units). The resultant ligation mixture wasused to transform E. coli JM109. From one of the transformants, thedesired plasmid pCC002A that is an expression vector for CC acylase N176was isolated and characterized by restriction mapping.

                  TABLE 1                                                         ______________________________________                                        Synthetic DNA oligomers for casette mutation of                               N-terminal of CC acylase N176(SEQ ID NO 57-63)                                restriction                                                                          sequence of synthetic DNA oligomers                                    sites of                                                                             upper strand: 5' → 3'                                                                            name/                                        each end                                                                             lower strand: 3' → 5'                                                                            length                                       ______________________________________                                        EcoRI/ AATTCGGATCCAAGCTTA        007a/18                                      MluI     GCCTAGGTTCGAATGCGC      007b/18                                                fMetThrMetAlaAlaAsnThr                                              ClaI/  CGATAAAATGACTATGGCGGCCAACACC                                                                            002a/28                                      Sau3AI  TATTTTACTGATACCGCCGGTTGTGGCTAG                                                                         002b/30                                      C1aI/     fMetThrMetAlaAlaAsnThr                                              BamHI  CGATAAAATGACTATGGCAGCTAATACG                                                                            013a/28                                              TATTTTACTGATACCGTCGATTATGCCTAG                                                                         013b/30                                      ______________________________________                                    

(2) Construction of pCK002, a kanamycin resistant expression vector forCC acylase N176

Plasmid pCC002A was digested with DraI (TOYOBO). The resultant mixturewas treated with phenol to remove the enzyme and precipitated by EtOH.The recovered DNA was suspended in 20 μl of a ligation buffer and mixedwith phosphorylated EcoRI linker (2 μg, Pharmacia), followed byincubation with T4 DNA ligase (300 units) at 4° C. for 16 hours. Thereaction mixture was extracted with phenol and precipitated by EtOH. Therecovered DNA was digested with EcoRI and the resultant 5.6 kb DNAlacking ampicillin resistant gene was isolated by agarose gelelectrophoresis. On the other hand, plasmid pA097 which is obtainablefrom a transformant Escherichia coli JM109 (pA097) FERM BP-37721 (1 μg)was digested with EcoRI, and the resulting 1.2 kb DNA of kanamycinresistance gene was isolated. The 1.2 kb EcoRI DNA was ligated to the5.6 kb EcoRI DNA with T4 DNA ligase (300 units) in 50 μl of a ligationbuffer at 16° C. for 2 hours. The ligation mixture was used to transformE. coli JM109 to obtain the desired plasmid pCK002 carrying kanamycinresistant gene for antibiotic marker.

EXAMPLE 3 (Construction of pCK013, a High Expression Vector for CCAcylase N176)

(1) Construction of pCC013A

(i) Construction of pCC007A

Plasmid pCC001A was digested with EcoRI and MluI and the resulting 6.4kb DNA fragment was isolated by agarose gel electrophoresis. Therecovered DNA was ligated to synthetic DNA oligomers 007a and 007b (0.5μg, respectively, Table 1), each of which were phosphorylated prior tothe ligation reaction, with T4 DNA ligase (300 units) at 16° C. for 5hours (SEQ ID NO:57,58). The resultant mixture was used to transform E.coli JM109 to obtain the desired plasmid pCC007A.

(ii) Construction of pCCNt013

Plasmid pCC007A (1.0 μg) was digested with ClaI and BamHI and theresultant 6.1 kb DNA was isolated by 5% polyacrylamide gelelectrophoresis. The DNA was ligated to synthetic oligomers 013a and013b (0.5 μg, respectively, each of which were phosphorylated, Table 1)with T4 DNA ligase (300 units) (SEQ ID NO:62,63). The ligation mixturewas used to transform E. coli JM109 and the desired plasmid pCCNt013 wasisolated from ampicillin resistant transformants.

(iii) Construction of pCC013A

Plasmid pCCNt013 was digested with BamHI and MluI and the resultant 6.1kb DNA was isolated. On the other hand, pCC002A (1.0 μg) was digestedwith MluI and Sau3AI to obtain 189 bp DNA fragment. The resultant DNAwas ligated to the 6.1 kb BamHI/MluI DNA fragment with T4 DNA ligase(300 units) and the ligation mixture was used to transform E. coliJM109. From one of the transformants resistant to ampicillin, thedesired plasmid pCC013A that has AT-rich NH₂ terminal DNA sequence(coding for the same amino acid sequence as that of native CC acylaseN176) was isolated.

(2) Construction of pCK013, a kanamycin resistant expression vector fornative CC acylase N176

(i) Construction of pΔN176

Plasmid pCK002 (1.0 μg) was digested with AatII (TOYOBO) and theresultant DNA was treated with T4 DNA ligase (150 units) forself-ligation. The ligation mixture was used to transform E. coli JM109to obtain the desired plasmid pΔN176 carrying a unique AtaII restrictionendonuclease site.

(ii) Construction of pCK013

Plasmid pΔN176 (1.0 μg) was digested with AatII and the linearized DNAwas treated with bacterial alkaline phosphatase (1 unit, Takara shuzo)in 100 mM Tris.HCl (pH 8.0) buffer 42° C. for 1 hour. Thedephosphorylated DNA was isolated and ligated to the 2.5 kb AatII DNAfrom pCC013A with T4 DNA ligase. The ligation mixture was used totransform E. coli JM109 to obtain the desired plasmid pCK013 carryingkanamaycin resistant gene for marker.

EXAMPLE 4 (Point Mutation of DNA Coding for CC Acylase N176 by Kunkel'sMethod)

(1) Subcloning of DNA coding for CC acylase N176 to M13 phage

(i) Preparation of mp18p181

Plasmid pCC013A (An expression vector for native CC acylase N176carrying ampicillin-resistant marker and construction of this plasmid isdisclosed in European Patent Application Publication No. 558,241, p.8)was digested with HpaI and Smal. The 842 bp DNA coding for Met¹ toPro²⁶⁷ of CC acylase N176 was isolated. The DNA was ligated to 7250 bpM13mp18 digested with SmaI in the presence of T4 DNA ligase and theligation mixture was used to transform E. coli JM109. From one of theplaques, the desired RF DNA mp18p181 in which the part of the acylaseDNA had been inserted in the reverse direction with plus ori of M13, wasisolated and characterized by restriction mapping. The phage solutionfrom which RF DNA mp18p181 was prepared was stored at 4° C. before use.

RF DNA mp18p183 was prepared from 1162 bp HpaI/Eco47III DNA coding forMet¹ to Ala⁴¹⁴ of CC acylase N176 from pCC013A and 7250 bp M13mp18digested with HincII in a manner similar to that described above.

(ii) Preparation of single-stranded U-mp18p181-SS(cf. Kunkel, T. A. etal. Methods Enzyml. 154, 367)

A single colony of E. coli CJ236 (dut-, ung-, F')(Bio-Rad Lab.) wascultured in 2 ml of 2XTY broth containing chloramphenicol (30 μg/ml) at37° C. for 16 hours. The cells (0.1 ml) were transferred to a fresh 2XTYbroth (50 ml) containing 30 μg/ml chloramphenicol and the cultivationwas continued at 37° C. When the absorbance at 600 nm reached 0.3, thephage solution (MOI<0.2) of mp18p181 was added to the culture. Thecultivation was continued for additional 5 hours. After centrifugationat 17,000×g at 4° C. for 15 min, the supernatant was centrifuged again.The resultant supernatant (30 ml) was treated with RNase (150 μg/ml,Sigma) at ambient temperature for 30 min, followed by addition of 7.5 mlof PEG solution (3.5M NH₄ OAc in 20% polyethyleneglycol 8,000). Aftercentrifugation (17,000×g, 15 min, 4° C.), the residue was suspended in200 μl of a buffer consisting of 300 mM NaCl, 100 mM Tris.HCl (pH 8.0)and 0.1 mM EDTA. The resultant solution was extracted with 200 μl ofphenol and 200 μl of phenol/CHCl₃ (1:1), successively, and washed twicewith CHCl₃ (200 μl). To the solution, 7.5M NH₄ OAc (100 μl ) and ethanol(600 μl) were added to precipitate phage DNA. The DNA was collected bycentrifugation, washed with 700 μl of ice-cooled 90% ethanol, and driedin vacuo. The purified single-stranded U-DNA (U-mp18p181-SS) wassuspended in 20 μl of TE buffer and stored at 4° C. before use.

Other single-stranded U-DNAs for Kunkel's mutation method were preparedin a manner similar to that described above.

(2) Preparation of mp18p181M164A for mutant CC acylase M164A

An oligodeoxyribonucleotide SO-M164A (0.2 nmol) was incubated with T4DNA kinase (10 units) in 15 μl of buffer consisting of 1.3 mM ATP, 10 mMdithiothreitol (DTT), 50 mM Tris.HCl, 6.6 mM in 5% polyethyleneglycol(PEG) 6,000 at 37° C. for 60 min (SEQ ID NO:2). The phosphorylatedprimer (1.5 μl, 20 pmol) was mixed with SS-U-DNA of mp18p181 (1 μl, 0.1pmol) in 20 μl of a buffer consisting of 10 mM Tris.HCl (pH 8.0), 6 mMand 40 mM NaCl. The mixture was heated at 75° C. for 5 min, followed bycooling to room temperature over 40 min, and then the mixture was placedat 0° C. To the mixture, T4 DNA polymerase (15 units), T4 DNA ligase(600 units), 100 mM dithiothreitol (DTT, 6 μl), 10 mM ATP (2 μl) and 5mM dNTP (dATP, dCTP, dGTP and dTTP, 2 μl ) were added. The resultingmixture was incubated at room temperature for 5 min and at 37° C. for1.5 h, successively. A portion of the reaction mixture (1.0 μl ) wasadded to competent cells (100 μl) of E. coli JM109 prepared according toSigesada's method Sigesada, K. (1983) SAIBO-KOUGAKU (Japanese) 2,616-626!, and the cells were incubated on wet ice for 30 min. To thetransformed cells, E. coli JM109 cultivated in L broth (A600=0.8, 200μl) was added, and the mixture of the cells was added to 3 ml of H Topagar (1% Bactotrypton, 0.8% NaCl, 0.8% agar) preheated at 55° C. Themixture was spread over an H plate (1% Bactotryptone, 0.8% NaCl, 1.5%agar) and the plate was incubated at 37° C. for 16 h. From plaques onthe plate, the desired RF DNA (mp18p181M164A) was isolated andcharacterized by digestion with BamHI.

(3) Preparation of pCKM164A for mutant CC acylase M164A

mp18p181M164A was digested with MluI and BstBI, and the 582 bp DNAfragment was isolated by agarose gel electrophoresis. Also, pCK013 (anexpression vector for native CC acylase N176 carryingkanamycin-resistant marker and construction of this plasmid is disclosedin European Patent Application Publication No. 558,241, p. 8) wasdigested with MluI and BstBI and a larger DNA fragment was isolated. Theresultant DNA (0.03 pmol) was ligated to the 582 bp MluI/BstBI DNA (0.15pmol) with T4 DNA ligase (300 units) in 10 μl of a ligation buffer (66mM Tris.HCl (pH 7.6), 6.6 mM, 10 mM β-mercaptoethanol, 0.5 mM ATP) atambient temperature for 5 hours. The ligation mixture was used totransform E. coli JM109. From one of the transformants resistant tokanamycin, the desired plasmid pCKM164A was isolated and characterizedby restriction mapping. The transformant was named as E. coliJM109/pCKM164A, a glycerol stock of which was prepared in a conventionalmanner.

(4) Preparation of mp18p181M174A for mutant CC acylase M174A

mp18p181M174A was prepared from SS-U-DNA of mp18p181 and DNA oligomerSO-M174A in a manner similar to that described above.

(5) Preparation of expression vector, pCKM174A for mutant CC acylaseM174A

An expression vector for mutant CC acylase M174A and a transformantthereof were prepared in a manner similar to that described above anddesignated as pCKM174A and E. coli JM109/pCKM174A, respectively. Aglycerol stock of the transformant was prepared in a conventionalmanner.

(6) Preparation of expression vector, pCKS166A for mutant CC acylaseS166A

mp18p183S166A for mutant CC acylase S166A was prepared from mp18p183 andDNA oligomer SO-S166A (5'-CATCCGCCAAAGCTTGAACCACACC GCACCCATAAG) in amanner similar to that described above. mp18p183S166A was digested withMluI and BstBI (SEQ ID NO:13). A smaller DNA fragment was isolated andligated with a larger DNA fragment of pCK013 digested with MluI andBstBI to give pCKS166A. E. coli JM109 was transformed with pCKS166A togive a transformant E. coli JM109/pCKS166A, a glycerol stock of whichwas prepared in a conventional manner.

EXAMPLE 5 (Point Mutation of DNA Coding for CC Acylase N176 by PCRMethod)

(1) Preparation of expression vector, pCKM164L for mutant CC acylaseM164L

pCK013 (template DNA, 0.5 fmol), DNA oligomer SO-MluFor primer #1, 125pmol, FIG. 1(b)! and SO-M164L primer #2, 125 pmol, FIG. 1(b)! were mixedwith Taq DNA polymerase (Kurabo, 1 unit) in 100 μl of a bufferconsisting of 10 mM Tris.HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 2.5mM and 0.2 mM dNTP (SEQ ID NO:15,25). The mixture was covered withmineral oil and PCR (Polymerase chain reaction) was carried out asfollows. After an initial denaturation (96° C. for 0.5 min), thereaction was performed for 30 cycles of amplification (97° C. for 1.5min, 50° C. for 2.5 min and 72° C. for 2.5 min), followed by finalextension (72° C. for 7 min). The resultant mixture was extracted withphenol, precipitated with ethanol and digested with BamHI and MluI. The285 bp BamHI/MluI DNA was ligated to a larger DNA fragment of pCKM164Adigested with BamHI and MluI. The ligation mixture was used to transformE. coli JM109. From one of the transformants resistant to kanamycin, thedesired plasmid pCKM164L was isolated and characterized by restrictionmapping. E. coli JM109 was transformed with pCKM164L to give atransformant E. coli JM109/pCKM164L, a glycerol stock of which wasprepared in a conventional manner.

(2) Preparation of expression vector, pCKM164G for mutant CC acylaseM164G

Mutation and amplification of the DNA fragment for mutant CC acylaseM164G was performed using pCK013 (template DNA) and DNA oligomersSO-MluFor primer #1, FIG. 1(b)! and SO-M164G FIG. 1(b)! in a mannersimilar to that described above. The resultant 285 bp BamHI/MluI DNA wasligated to a larger DNA fragment of pCKM164A digested with MluI andBamHI to give pCKM164G (SEQ ID NO:15,21). E. coli JM109 was transformedwith pCKM164G (SEQ ID NO:15,21) to give a transformant E. coliJM109/pCKM164G, a glycerol stock of which was prepared in a conventionalmanner.

(3) Expression vectors for other M164 mutant CC acylase

Expression vectors for M164X mutant acylases (X=C, D, E, F, H, I, K, N,P, Q, R, S, T, V, W or Y) and transformants thereof were prepared in amanner similar to that described above.

EXAMPLE 6 (Preparation of Expression Vectors for Other Mutant CCAcylases)

(1) Construction of mp19pfu62

M13mp19 (1.0 μg) was digested with SmaI (5 units) and HindIII (5 units)and the resulting 7.2 kb DNA was isolated by agarose gelelectrophoresis. On the other hand, pCK002 (construction of this plasmidis disclosed in European Patent Application Publication No. 558,241, p.7) was digested with SmaI and HindIII and a 1.6 kb DNA was isolated. Theresulting DNA was ligated to the 7.2 kb SmaI/HindIII DNA with T4 DNAligase (300 units) in 20 μl of a ligation buffer at 15° C. for 2 h. Theligation mixture was used to transform E. coli JM109 to obtain thedesired RF DNA mp19pfu62. The single-stranded U-DNA (SS-U-DNA) ofmp19pfu62 was prepared in a manner similar to that described in Example2 (1)(ii).

(2) Preparation of mp19pfu62M465A for mutant CC acylase M465A

mp19pfu62M465A was prepared from SS-U-DNA of mp19pfu62 and DNA oligomerSO-M465A FIG. 1(a)! in a manner similar to that described above (SEQ IDNO:6).

(3) Preparation of mp19pfu62M506A for mutant CC acylase M506A

mp19pfu62M506A was prepared from SS-U-DNA of mp19pfu62 and DNA oligomerSO-M506A FIG. 1(a)! in a manner similar to that described above (SEQ IDNO:8).

(4) Preparation of mp19pfu62M750A for mutant CC acylase M750A

mp19pfu62M750A was prepared from SS-U-DNA of mp19pfu62 and DNA oligomerSO-M750A FIG. 1(a)! in a manner similar to that described above (SEQ IDNO:10).

(5) Preparation of expression vector, pCKM465A for mutant CC acylaseM465A

mp19pfu62M465A was digested with PstI and NcoI, and a smaller DNAfragment (1271 bp) was isolated by agarose gel electrophoresis. Also,pCK013 was digested with PstI and NcoI. A larger DNA was isolated andligated to the 1271 bp PstI/NcoI DNA with T4 DNA ligase in a bufferconsisting of 50 mM Tris.HCl, 10 mM, 1.0 mM DTT and 5% PEG 6,000. Theligation mixture was used to transform E. coli DH10B. From one of thetransformants resistant to kanamycin, the desired plasmid pCKM465A wasisolated and characterized by restriction mapping. E. coli JM109 wastransformed with pCKM465A to give a transformant E. coli JM109/pCKM465A,a glycerol stock of which was prepared in a conventional manner.

(6) Preparation of expression vector, pCKM506A for mutant CC acylaseM506A

The pCKM506A was constructed from pCK013 and mp19pfu62M506A in a mannersimilar to that described above. E. coli JM109 was transformed withpCKM506A to give a transformant E. coli JM109/pCKM506A, a glycerol stockof which was prepared in a conventional manner.

(7) Preparation of expression vector, pCKM750A for mutant CC acylaseM750A

The pCKM750A was constructed from pCK013 and mp19pfu62M750A in a mannersimilar to that described above. E. coli JM109 was transformed withpCKM750A to give a transformant E. coli JM109/pCKM750A, a glycerol stockof which was prepared in a conventional manner.

EXAMPLE 7 (Preparation of Expression Vectors for E358 Mutant CCAcylases)

(1) Preparation of expression vectors for M269I/E358 mutant CC acylases

(i) Preparation of expression vector, p269I358K for mutant CC acylaseM269I/E358K

A larger DNA fragment of pCK013 digested with HpaI and MluI (0.5 fmol),DNA oligomer SO-E358K 5'-TAACCGGGCCATGGCGCGTCTTGACTATAT CGAACT, 125pmol, listed in FIG. 1(b)(ii)! and SO-BstFor5'-ATCGCGTCTTCGAAATACCGGGCATC, 125 pmol, listed in FIG. 1(b)(ii)! weremixed with Taq DNA polymerase (TaKaRa, 1 unit) in 100 μl of a bufferconsisting of 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 0.1% gelatin, 1.5 mMand 0.2 mM dNTP (SEQ ID NO:38,36). The mixture was covered with mineraloil and subjected to initial denaturation (96° C. for 0.5 min), 25cycles of amplification (97° C. for 1.5 min, 50° C. for 2.5 min and 72°C. for 2.5 min.) and final extension (72° C. for 7 min). The reactionmixture was extracted with phenol, precipitated with ethanol, anddigested with NcoI and BstBI. The resultant DNA (290 bp) was ligated toa larger DNA fragment of pCK013 digested with NcoI and BstBI. Theligation mixture was used to transform E. coli DH10B (purchased fromGibco-BRL). From one of the transformants resistant to kanamycin, thedesired plasmid p269I358K was isolated and characterized by restrictionmapping. E. coli JM109 was transformed with p269I358K to give atransformant E. coli JM109/p269I358K, a glycerol stock of which wasprepared in a conventional manner.

(ii) Preparation of expression vectors, p269I358S and p269I358L formutant CC-acylases M269I/E358S and M269I/E358L, respectively

Expression vector for M269I/E358S (or M269I/E358L) was prepared frompCK013 and DNA oligomers SO-BstFor and SO-E358S or SO-E358L, listed inFIG. 1(b)(ii)! in a manner similar to that described above (SEQ IDNO:41,37). E. coli JM109 was transformed with p269I358S (or p269I358L)to give a transformant E. coli JM109/p269I358S (or E. coliJM109/p269I358L), a glycerol stock of which was prepared in aconventional manner.

(2) Preparation of expression vectors for other E358 mutant CC acylases

(i) Preparation of expression vector, pCCE358R for mutant CC acylaseE358R

mp18p183E358R for mutant CC acylase E358R was prepared from mp18p183 andDNA oligomers SO-E358R FIG. 1(b)(ii)! in a manner similar to thatdescribed in Example 2 (SEQ ID NO:40). A smaller DNA of mp18p183E358Rdigested with MluI and NcoI was ligated to a larger DNA of pCC013Adigested with MluI and NcoI to give pCCE358R. E. coli JM109 wastransformed with pCCE358R to give a transformant E. coli JM109/pCCE358R,a glycerol stock of which was prepared in a conventional manner.

(ii) Preparation of expression vector, mutant CC acylase pCCE358T forE358T

mp18p183E358T for mutant CC acylase E358T was prepared from mp18p183 andDNA oligomer SO-E358T FIG. 1(b)(ii)! in a manner similar to thatdescribed in Example 2 (SEQ ID NO:42). A smaller DNA of mp18p183E358Tdigested with MluI and-NcoI was ligated to a larger DNA of pCC013Adigested with MluI and NcoI to give pCCE358T. E. coli JM109 wastransformed with pCCE358T to give a transformant E. coli JM109/pCCE358T,a glycerol stock of which was prepared in a conventional manner.

EXAMPLE 8 (Preparation of Multiple Mutant CC Acylases)

(1) Combination of M164L or M164A with another mutant CC acylase

(i) Preparation of expression vector, p164L269Y for mutant CC acylaseM164L/M269Y

pCKM164L was digested with MluI and BstBI and a smaller DNA (582 bp) wasisolated. On the other hand, pCKM269Y (construction method of thisplasmid is disclosed in European Patent Application Publication No.558,241, p. 10) was digested with MluI and BstBI. The resultant DNA (ca6.2 kb) was isolated and ligated to 582 bp MluI/BstBI DNA. The ligationmixture was used to transform E. coli DH10B. From one of thetransformants resistant to kanamycin, the desired p164L269Y was isolatedand characterized by restriction mapping. E. coli JM109 was transformedwith p164L269Y to give a transformant E. coli JM109/p164L269Y, aglycerol stock of which was prepared in a conventional manner.

(ii) Preparation of expression vector, p164L269F for mutant CC acylaseM164L/M269F

pCKM164L was digested with MluI and BstBI and a smaller DNA (582 bp) wasisolated. On the other hand, pCKM269F (construction method of thisplasmid is disclosed in European Patent Application Publication No.558,241, p. 15-16) was digested with MluI and BstBI. A larger DNAfragment (ca 6.2 kb) was isolated and ligated to 582 bp MluI/BstBI DNA.The ligation mixture was used to transform E. coli DH10B. From one ofthe transformants resistant to kanamycin, the desired p164L269F wasisolated and characterized by restriction mapping. E. coli JM109 wastransformed with p164L269F to give a transformant E. coliJM109/p164L269F, a glycerol stock of which was prepared in aconventional manner.

(iii) Preparation of expression vector, p164L269Y305S for mutant CCacylase M164L/M269Y/C305S

p164L269Y305S was prepared from pCKM164L and p269Y305S (constructionmethod of this plasmid is disclosed in European Patent ApplicationPublication No. 558,241, p. 10) in a manner similar to that describedabove. E. coli JM109 was transformed with p164L269Y305S to give atransformant E. coli JM109/p164L269Y305S, a glycerol stock of which wasprepared in a conventional manner.

(iv) Preparation of expression vector, p164L174A for mutant CC acylaseM164L/M174A

HpaI/NcoI DNA (1122 bp) from pCKM164L (template DNA, 0.5 fmol), DNAoligomers SO-MluFor primer #1, 125 pmol, FIG. 1(b)(i)! and SO-M174A2(5'-GACCGGCAGCGCTAGCGCCCGCCAGAGCTTGA, primer #2, 125 pmol) were mixedwith Taq DNA polymerase (TaKaRa, 1 unit) in 100 μl of a bufferconsisting of 10 mM Tris.HCl (pH 8.3), 50 mM KCl, 0.1% gelatin, 1.5 mMand 0.2 mM dNTP (SEQ ID NO:64). The mixture was covered with mineral oiland subjected to initial denaturation (96° C. for 0.5 min), 25 cycles ofamplification (97° C. for 1.5 min, 50° C. for 2.5 min and 72° C. for 2.5min) and final extension (72° C. for 7 min). The resultant mixture wasextracted with phenol, precipitated with ethanol, and digested with MluIand NheI. The resultant DNA was ligated to a larger DNA fragment ofpCKM174A digested with MluI and NheI to give the desired plasmidp164L174A. E. coli JM109 was transformed with the p164L174A to give atransformant E. coli JM109/p164L174A, a glycerol stock of which wasprepared in a conventional manner.

(v) Preparation of expression vector, p164A174A for mutant CC acylaseM164A/M174A

p164A174A was prepared from pCKM164A (template DNA), SO-MluFor primer#1, FIG. 1(b)(i)!, SO-M174A primer #2, FIG. 1(a)! and pCKM174A (vectorDNA) in a manner similar to that described above (SEQ ID NO:15,14). E.coli JM109 was transformed with p164A174A to give a transformant E. coliJM109/p164A174A, a glycerol stock of which was prepared in aconventional manner.

(vi) Preparation of expression vector, p164A269Y for mutant CC acylaseM164A/M269Y

M164A was digested with MluI and BstBI and a small DNA (582 bp) wasisolated. On the other hand, pCKM269Y was digested with MluI and BstBI.The resultant larger DNA fragment was ligated to the 582 bp MluI/BstBIDNA and the ligation mixture was used to transform E. coli DH10B. Fromone of the transformants resistant to kanamycin, the desired plasmidp164A269Y was isolated and characterized by restriction mapping. E. coliJM109 was transformed with p164A269Y to give a transformant E. coliJM109/p164A269Y, a glycerol stock of which was prepared in aconventional manner.

(vii) Preparation of expression vectors for mutant CC acylases,M164L/M174A/M269Y, M164L/M174A/M269F and M164A/M174A/M269Y/C305S

A smaller DNA of p164L174A digested with MluI and BstBI was ligated to alarger DNA fragment of pCKM269Y (or pCKM269F or p269Y305S) digested withMluI and BstBI to give the desired vector, p164L174A269Y (orp164L174A269F or p164A174A269Y305S). E. coli JM109 was transformed withthe p164L174A269Y (or p164L174A269F or p164A174A269Y305S) to give atransformant E. coli JM109/p164L174A269Y (or E. coli JM109/p164L174A269For E. coli JM109/p164A174A269Y305S), a glycerol stock of which wasprepared in a conventional manner.

(viii) Preparation of expression vector, p164L174A269Y305S750A formutant CC acylase, M164L/M174A/M269Y/C305S/M750A

pCKM750A was digested with PstI and NcoI. A smaller DNA was isolated andligated to the larger DNA of p164L174A269Y305S digested with PstI andNcoI to give the desired plasmid. E. coli JM109 was transformed withp164L174A269Y305S750A to give a transformant E. coliJM109/p164L174A269Y305S750A, a glycerol stock of which was prepared in aconventional manner.

(2) Combination of A49L with other mutant acylases

(i) Preparation of mp18p181A49L for mutant CC acylase A49L

mp18p181A49L was prepared from SS-U-DNA of mp18p181 and DNA oligomerSO-A49L in a manner similar to that described in Example 2 (SEQ IDNO:12).

(ii) Preparation of expression vector, pCKA49L for mutant CC acylaseA49L

mp18p181A49L was digested with ClaI and MluI and a 218 bp DNA wasisolated. On the other hand, pCC013A was digested with ClaI and MluI.The resultant larger DNA was isolated and ligated to the 218 bpClaI/MluI DNA. The ligation mixture was used to transform E. coli JM109.From one of the transformants resistant to ampicillin, an expressionvector for mutant CC acylase A49L, designated as pCCA49L, was isolatedand characterized by restriction mapping. A 250 bp HpaI/MluI DNAfragment from pCCA49L was ligated to a larger DNA fragment of pCK013digested with HpaI and MluI to give pCKA49L.

(iii) Comparison of expression of native CC acylase and mutant CCacylase A49L

Glycerol stock solution (1 ml) of E. coli JM109/pCKA49L (orJM109/pCK013) which had been prepared by transforming E. coli JM109 withthe plasmid pCKA49L (or pCK013) in a conventional manner was transferredto 100 ml of L broth containing 50 μg/ml kanamycin, and the mixture wascultured at 30° C. for 8 hours. The cultured broth (3.75 ml) was addedto 25 ml of N-3 broth (ingredients: 5% soybean sauce, 1% glycerol, 1.25%K₂ HPO₄, 0.38% KH₂ PO₄, 50 μg/ml thiamine.HCl, 2 mM MgSO₄.7H₂ O₄ 0.2 mMCaCl₂.2H₂ O, 0.05 mM FeSo₄.7H₂ O) containing 25 μg/ml kanamycin, and themixture was cultured at 22.5° C. for 16 hours. At 16 h, 3-indoleacrylicacid (IAA) was added to the cultured broth to a final concentration of20 μg/ml and the cultivation was continued for additional 56 hours.Cells were harvested by centrifugation at 14,000 rpm for 15 min at 4°C., suspended in 40 ml of TE buffer (pH 8.0) and lysed by sonication.The lysate was centrifuged at 14,000 rpm for 20 min at 4° C. to obtainthe supernatant (designated as "soup" fraction). The residues wereresuspended in 40 ml of a buffer containing 100 mM Tris.HCl (pH 8.0), 1mM EDTA and 8M urea and lysed by sonication. After centrifugation toremove insoluble materials, the supernatant was collected (designated as"ppt" fraction). The "soup" and "ppt" fractions of mutant CC acylaseA49L and native CC acylase N176 were analyzed by 15% SDS-PAGE. Thecellular insoluble precursor protein was greatly decreased by mutationfrom a native CC acylase to a mutant CC acylase A49L. The resultscorresponded to the amounts of mature acylases (native CC acylase ormutant CC acylase A49L) in "soup" assayed by reversed phase HPLC (in thefollowing Table).

    ______________________________________                                        A49L (units/ml broth)                                                                         native (units/ml broth)                                       ______________________________________                                        72.6            35.6                                                          ______________________________________                                    

(iv) Preparation of expression vector, p49L164L174A269Y for mutant CCacylase, A49L/M164L/M174A/M269Y

The 772 bp MluI/NcoI DNA from p164L174A269Y was ligated to a larger DNAof pCKA49L digested with MluI and NcoI to give the desired plasmidp49L164L174A269Y. E. coli JM109 was transformed with p49L164L174A269Y togive a transformant E. coli JM109/p49L164L174A269Y, a glycerol stock ofwhich was prepared in a conventional manner.

EXAMPLE 9 (Expression and Purification of Mutant CC Acylases)

(1) Expression of mutant CC acylase M164A

A glycerol stock of E. coli JM109/pCKM164A (0.5 ml) was added to 50 mlof L broth containing 50 μg/ml kanamycin and the mixture was cultivatedat 30° C. for 8 h. The cultivated broth (3.75 ml) was transferred to 25ml of N-3 broth (5.0% soybean source (Osaka Shokuhinn Kagaku), 0.608%Na₂ HPO₄, 0.7% KH₂ PO₄, 0.7% K₂ HPO₄, 0.12% (NH₄)₂ SO₄, 0.02% NH₄ Cl,0.0011% FeSO₄.7H₂ O, 0.0011% CaCl₂.2H₂ O, 0.000276% MnSO₄.nH₂ O,0.000276% AlCl₃.6 H₂ O, 0.00011% CoCl₂.6H₂ O, 0.0000552% ZnSO₄.7H₂ O,0.0000552% NaMoO₄.2H₂ O, 0.0000276% CuSO₄.7H₂ O, 0.0000138% H₃ BO₄, 50μg/ml vitamin B1, 0.048% MgSO₄) containing 1.0% glycerol and 12.5 μg/mlkanamycin. The mixture was incubated at 20°-22° C. for 88 h withaddition of β-indoleacrylic acid (final concentration 20 μg/ml) at 16 hand glycerol (final concentration 1.0%) at 16 and 24 h after the startof cultivation. Cells were harvested by centrifugation (8,000 rpm at 4°C. for 10 min), suspended in 10 ml of TE buffer (10 mM Tris.HCl (pH8.0), 1.0 mM EDTA) and lysed by sonication. After centrifugation (15,000rpm at 4° C. for 20 min), the supernatant (crude lysate) was stored at4° C. until use.

(2) Purification of M164A

The crude lysate (1.0 ml) was filtrated using a 0.45 μm column guard(Millipore) and applied to a TSK-gelTM DEAE-5PW (TOSOH, 4.6×50 mm) highperformance liquid chromatography column. Elution was performed with aconcave gradient from 80% of A buffer 25 mM Tris.HCl (pH 8.0)!+20% of Bbuffer 0.5M NaCl-25 mM Tris.HCl (pH 8.0)! to 50% of A buffer+50% of Bbuffer over 30 min at a flow rate of 1.0 ml/min. Absorbance at 230 nmwas used to monitor the eluate. The last main peak eluted withapproximately 0.16M NaCl was collected to obtain a pure preparation ofmutant CC acylase M164A.

(3) Expression of other mutant CC acylases

Cultivation of E. coli JM109 carrying another expression vector (such aspCKM164L, pCKM164G, pCKM174A, pCKM465A, pCKM506A, pCKM750A, p164L/269Y,p164L/269Y/305S, p164L/174A/269Y/305S, p269I/358K, p269I/358S,p164L/269F, p164L/174A/269F, pCKS166A, p164L/174A/269Y/305S/750A,p164A/269Y, pCK49L and p49L/164L/174A) and preparation of the crudelysate was performed in a manner similar to that described above.

(4) Purification of other mutant CC acylases

Other mutant CC acylases (such as M164L, M164G, M174A, M465A, M506A,M750A, M164L/M269Y, M164L/M269Y/C305S, M164L/M174A/M269Y/S305S,M269I/E358K, M269I/E358S, M164L/M269F, M164L/M174A/M269F, S166A,M164L/M174A/M269Y/C305S/M750A, M164A/269Y, and A49L/M164L/M174A) werepurified from each of crude lysates in a manner similar to thatdescribed above.

(5) Identification of mutant CC acylases

Purified mutant CC acylases such as M164A, M164L, M164G, M174A, M465A,M506A, M750A, M164L/M269Y, M164L/M269Y/C305S, M164L/M174A/M269Y/S305S,M269I/E358K, M269I/E358S, M164L/M269F, M164L/M174A/M269F, S166A,M164L/M174A/M269Y/C305S/M750A, M164A/269Y, and A49L/M164L/M174A werecharacterized by 12.5% SDS-PAGE analysis and reversed phase HPLC. Fromthe SDS-PAGE analysis in the presence of β-mercaptoethanol, eachpurified acylase was confirmed to consist of two independent subunits,25.4 kDa and 58.4 kDa peptides corresponding to α and β subunits,respectively, whose molecular weights were calculated from theirmobility on gel electrophoresis. In HPLC analysis, each purified acylasewas dissociated to 2 independent peptides, α and β subunits, which wereeluted at approximately 8.7 and 5.8 min, respectively HPLC conditions,column: 5C4-AR-300, 4.6×50 mm; eluate: linear gradient from 35% to 70%aqueous acetonitrile containing 0.05% trifluoroacetic acid over 10 min;detection: 214 nm!. The both subunits of each mutant acylase wereisolated by the reversed phase HPLC system and determined to beidentical to the sequence of native acylase by amino terminal sequenceanalysis with 473A protein sequencer (Applied Biosystems).

EXAMPLE 10 (DNA Sequence Analysis)

DNA sequence of vectors for mutant acylases such as M164A, M164L, M164G,M174A, M465A, M506A, M750A, M164L/M269Y, M164L/M269Y/C305S,M164L/M174A/M269Y/S305S, M269/E358K, M269I/E358S, M164L/M269F,M164L/M174A/M269F, S166A, M164L/M174A/M269Y/C305S/M750A, M164A/269Y, andA49L/M164L/M174A was determined by 373A DNA sequencer (AppliedBiosystems) and confirmed to be identical to that as expected.

EXAMPLE 11 (CC Acylase Activity)

The CC acylase activity at pH 8.7 of each of the mutant acylases aslisted in Table 2 was determined in the same manner as described above.The results are shown in Table

                  TABLE 2                                                         ______________________________________                                        Relative activity of CC acylase:                                              mutant acylase       CC acylase activity                                      ______________________________________                                        native (N176)         100*                                                    M164L                122                                                      M174A                123                                                      M465A                136                                                      M506A                140                                                      M750A                142                                                      M164L/M269Y          161                                                      M164L/M269Y/C305S    141                                                      M164L/M174A/M269Y/C305S                                                                            155                                                      M269I/E358K          153                                                      M269I/E358S          184                                                      M164L/M269F          193                                                      M164L/M174A/M269F    184                                                      S166A                186                                                      M164L/M174A/M269Y    245                                                      M164L/M174A/M269Y/C305S/M750A                                                                      192                                                      M164A/M269Y          172                                                      A49L/M164L/M174A/M269Y                                                                             226                                                      ______________________________________                                         (*: calculated as native = 100%)                                         

EXAMPLE 12 (GL-7ACA Acylase Activity)

The GL-7ACA acylase activity at pH 7.5 of each of the mutant acylases aslisted in Table 3 was determined in the same manner as described above.The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Relative activity of GL-7ACA acylase:                                         mutant acylase                                                                             GL-7ACA acylase activity                                         ______________________________________                                        native (N176)                                                                               100*                                                            M164A        167                                                              M164G        162                                                              ______________________________________                                         (*: calculated as native = 100%)                                         

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 64                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlyLeuLeuAlaGlySerValTrpPhe                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GGGCCTGCTTGCGGGATCCGTGTGGTTCA29                                               (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       LeuTrpArgAlaLeuAlaLeuPro                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GCTCTGGCGGGCGCTAGCGCTGCCGG26                                                  (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       HisGluAlaAlaProArgValIle                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GCACGAGGCGGCGCCACGCGTGATCG26                                                  (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GluArgIleAlaLysArgLeuValAla                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       GAGCGCATCGCGAAGCGCTTGGTCGCCA28                                                (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AspCysAlaAlaValProMetLeu                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CGACTGTGCGGCGGTACCGATGCTCT26                                                  (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AlaSerGlyGluLeuAspAlaTyrArg                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GCCTCGGGCGAGCTCGATGCCTATCGG27                                                 (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CATCCGCCAAAGCTTGAACCACACCGCACCCATAAG36                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      MetGluLeuThrArgArgLysAlaLeuGlyArg                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      ATGGAGCTGACGCGTCGCAAAGCGCTGGGACG32                                            (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      ValMetArgArgLeuGlyLeuLeuCysGlySerVal                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CGGTGATGCGTCGACTCGGCCTGCTTTGCGGATCCGTG38                                      (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      CACGGATCCATCAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CACGGATCCTTCAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      CACGGATCCGAAAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      CACGGATCCGCCAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      CACGGATCCATGAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CACGGATCCGATAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      CACGGATCCCTTAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CACGGATCCCAGAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      CACGGATCCGTTAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CACGGATCCCGGAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      CACGGATCCCTGAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      CACGGATCCACGAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CACGGATCCCGAAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CACGGATCCCGTAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      CACGGATCCCACAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      CACGGATCCCCAAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CACGGATCCCACAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      ArgValPheGluIleProGlyIle                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 38 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      CACGGATCCATAAAGCAGGCCGAGTCGACGCATCACCG38                                      (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 11 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      PheAspIleValLysThrArgHisGlyProVal                                             1510                                                                          (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TCAAGCTATATCAGTTCTGCGCGGTACCGGGCCAAT36                                        (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      TAACCGGGCCATGGCGCGTCAGGACTATATCGAACT36                                        (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      TAACCGGGCCATGGCGCGTGCGGACTATATCGAACT36                                        (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      TAACCGGGCCATGGCGCGTCGAGACTATATCGAACT36                                        (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      TAACCGGGCCATGGCGCGTGGTGACTATATCGAACT36                                        (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2325 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..2325                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 4..2322                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      ATGACTATGGCAGCTAATACGGATCGCGCGGTCTTGCAGGCGGCGCTG48                            MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       CCGCCGCTTTCCGGCAGCCTCCCCATTCCCGGATTGAGCGCGTCGGTC96                            ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        CGCGTCCGGCGCGATGCCTGGGGCATCCCGCATATCAAGGCCTCGGGC144                           ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GAGGCCGATGCCTATCGGGCGCTGGGCTTCGTCCATTCGCAGGACCGT192                           GluAlaAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        CTTTTCCAGATGGAGCTGACGCGTCGCAAGGCGCTGGGACGCGCGGCC240                           LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GAATGGCTGGGCGCCGAGGCCGCCGAGGCCGATATCCTCGTGCGCCGG288                           GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      CTCGGAATGGAAAAAGTCTGCCGGCGCGACTTCGAGGCCTTGGGCGTC336                           LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GAGGCGAAGGACATGCTGCGGGCTTATGTCGCCGGCGTGAACGCATTC384                           GluAlaLysAspMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     CTGGCTTCCGGTGCTCCCCTGCCTGTCGAATACGGATTGCTCGGAGCA432                           LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GAGCCGGAGCCCTGGGAGCCTTGGCACAGCATCGCGGTGATGCGCCGG480                           GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     CTGGGCCTGCTTGCGGGATCCGTGTGGTTCAAGCTCTGGCGGATGCTG528                           LeuGlyLeuLeuAlaGlySerValTrpPheLysLeuTrpArgMetLeu                              160165170175                                                                  GCGCTGCCGGTGGTCGGAGCCGCGAATGCGCTGAAGCTGCGCTATGAC576                           AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     GATGGCGGCCGGGATTTGCTCTGCATCCCGCCGGGCGCCGAAGCCGAT624                           AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     CGGCTCGAGGCGGATCTCGCGACCCTGCGGCCCGCGGTCGATGCGCTG672                           ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     CTGAAGGCGATGGGCGGCGATGCCTCCGATGCTGCCGGCGGCGGCAGC720                           LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AACAACTGGGCGGTCGCTCCGGGCCGCACGGCGACCGGCAGGCCGATC768                           AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  CTCGCGGGCGATCCGCATCGCGTCTTCGAAATCCCGGGCATGTATGCG816                           LeuAlaGlyAspProHisArgValPheGluIleProGlyMetTyrAla                              260265270                                                                     CAGCATCATCTGGCCTGCGACCGGTTCGACATGATCGGCCTGACCGTG864                           GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     CCGGGCGTGCCGGGCTTCCCGCACTTCGCGCATAACGGCAAGGTCGCC912                           ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TATTGCGTCACCCATGCCTTCATGGACATCCACGATCTCTATCTCGAG960                           TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     CAGTTCGCGGGGGAGGGCCGCACTGCGCGGTTCGGCAACGATTTCGAG1008                          GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  CCCGTCGCCTGGAGCCGGGACCGTATCGCGGTCCGGGGTGGCGCCGAT1056                          ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     CGCGAGTTCGATATCGTCGAGACGCGCCATGGCCCGGTTATCGCGGGC1104                          ArgGluPheAspIleValGluThrArgHisGlyProValIleAlaGly                              355360365                                                                     GATCCGCGCGATGGCGCAGCGCTCACGCTGCGTTCGGTCCAGTTCGCC1152                          AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GAGACCGATCTGTCCTTCGACTGCCTGACGCGGATGCCGGGCGCATCG1200                          GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ACCGTGGCCCAGCTCTACGACGCGACGCGCGGCTGGGGCCTGATCGAC1248                          ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  CATAACCTCGTCGCCGGGGATGTCGCGGGCTCGATCGGCCATCTGGTC1296                          HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     CGCGCCCGCGTTCCGTCCCGTCCGCGCGAAAACGGCTGGCTGCCGGTG1344                          ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     CCGGGCTGGTCCGGCGAGCATGAATGGCGGGGCTGGATTCCGCACGAG1392                          ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     GCGATGCCGCGCGTGATCGATCCGCCGGGCGGCATCATCGTCACGGCG1440                          AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AATAATCGCGTCGTGGCCGATGACCATCCCGATTATCTCTGCACCGAT1488                          AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  TGCCATCCGCCCTACCGCGCCGAGCGCATCATGAAGCGCCTGGTCGCC1536                          CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AATCCGGCTTTCGCCGTCGACGATGCCGCCGCGATCCATGCCGATACG1584                          AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     CTGTCGCCCCATGTCGGGTTGCTGCGCCGGAGGCTCGAGGCGCTTGGA1632                          LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     GCCCGCGACGACTCCGCGGCCGAAGGGCTGAGGCAGATGCTCGTCGCC1680                          AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TGGGACGGCCGCATGGATGCGGCTTCGGAGGTCGCGTCTGCCTACAAT1728                          TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  GCGTTCCGCAGGGCGCTGACGCGGCTGGTGACGGACCGCAGCGGGCTG1776                          AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GAGCAGGCGATATCGCATCCCTTCGCGGCTGTCGCGCCGGGCGTCTCA1824                          GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     CCGCAAGGCCAGGTCTGGTGGGCCGTGCCGACCCTGCTGCGCGACGAC1872                          ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     GATGCCGGAATGCTGAAGGGCTGGAGCTGGGACCAGGCCTTGTCTGAG1920                          AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     GCCCTCTCGGTCGCGTCGCAGAACCTGACCGGGCGAAGCTGGGGCGAA1968                          AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GAGCATCGGCCGCGCTTCACGCATCCGCTTGCCACGCAATTCCCGGCC2016                          GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TGGGCGGGGCTGCTGAATCCGGCTTCCCGTCCGATCGGTGGCGATGGC2064                          TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     GATACCGTGCTGGCGAACGGGCTCGTCCCGTCAGCCGGGCCGCAGGCG2112                          AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ACCTATGGTGCCCTGTCGCGCTACGTCTTCGATGTCGGCAATTGGGAC2160                          ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AATAGCCGCTGGGTCGTCTTCCACGGCGCCTCCGGGCATCCGGCCAGC2208                          AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  GCCCATTATGCCGATCAGAATGCGCCCTGGAGCGACTGTGCGATGGTG2256                          AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     CCGATGCTCTATAGCTGGGACAGGATCGCGGCAGAGGCCGTGACGTCG2304                          ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     CAGGAACTCGTCCCGGCCTGA2325                                                     GlnGluLeuValProAla*                                                           770                                                                           (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 774 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GluAlaAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GluAlaLysAspMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     LeuGlyLeuLeuAlaGlySerValTrpPheLysLeuTrpArgMetLeu                              160165170175                                                                  AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  LeuAlaGlyAspProHisArgValPheGluIleProGlyMetTyrAla                              260265270                                                                     GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     ArgGluPheAspIleValGluThrArgHisGlyProValIleAlaGly                              355360365                                                                     AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     GlnGluLeuValProAla                                                            770                                                                           (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2325 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..2325                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 4..2322                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      ATGACTATGGCAGCTAATACGGATCGCGCGGTCTTGCAGGCGGCGCTG48                            MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       CCGCCGCTTTCCGGCAGCCTCCCCATTCCCGGATTGAGCGCGTCGGTC96                            ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        CGCGTCCGGCGCGATGCCTGGGGCATCCCGCATATCAAGGCCTCGGGC144                           ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GAGGCCGATGCCTATCGGGCGCTGGGCTTCGTCCATTCGCAGGACCGT192                           GluAlaAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        CTTTTCCAGATGGAGCTGACGCGTCGCAAGGCGCTGGGACGCGCGGCC240                           LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GAATGGCTGGGCGCCGAGGCCGCCGAGGCCGATATCCTCGTGCGCCGG288                           GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      CTCGGAATGGAAAAAGTCTGCCGGCGCGACTTCGAGGCCTTGGGCGTC336                           LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GAGGCGAAGGACATGCTGCGGGCTTATGTCGCCGGCGTGAACGCATTC384                           GluAlaLysAspMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     CTGGCTTCCGGTGCTCCCCTGCCTGTCGAATACGGATTGCTCGGAGCA432                           LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GAGCCGGAGCCCTGGGAGCCTTGGCACAGCATCGCGGTGATGCGCCGG480                           GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     CTGGGCCTGCTTATGGGTGCGGTGTGGTTCAAGCTTTGGCGGATGCTG528                           LeuGlyLeuLeuMetGlyAlaValTrpPheLysLeuTrpArgMetLeu                              160165170175                                                                  GCGCTGCCGGTGGTCGGAGCCGCGAATGCGCTGAAGCTGCGCTATGAC576                           AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     GATGGCGGCCGGGATTTGCTCTGCATCCCGCCGGGCGCCGAAGCCGAT624                           AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     CGGCTCGAGGCGGATCTCGCGACCCTGCGGCCCGCGGTCGATGCGCTG672                           ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     CTGAAGGCGATGGGCGGCGATGCCTCCGATGCTGCCGGCGGCGGCAGC720                           LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AACAACTGGGCGGTCGCTCCGGGCCGCACGGCGACCGGCAGGCCGATC768                           AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  CTCGCGGGCGATCCGCATCGCGTCTTCGAAATCCCGGGCATGTATGCG816                           LeuAlaGlyAspProHisArgValPheGluIleProGlyMetTyrAla                              260265270                                                                     CAGCATCATCTGGCCTGCGACCGGTTCGACATGATCGGCCTGACCGTG864                           GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     CCGGGCGTGCCGGGCTTCCCGCACTTCGCGCATAACGGCAAGGTCGCC912                           ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TATTGCGTCACCCATGCCTTCATGGACATCCACGATCTCTATCTGGAG960                           TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     CAGTTCGCGGGGGAGGGCCGCACTGCGCGGTTCGGCAACGATTTCGAG1008                          GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  CCCGTCGCCTGGAGCCGGGACCGTATCGCGGTCCGGGGTGGCGCCGAT1056                          ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     CGCGAGTTCGATATCGTCGAGACGCGCCATGGCCCGGTTATCGCGGGC1104                          ArgGluPheAspIleValGluThrArgHisGlyProValIleAlaGly                              355360365                                                                     GATCCGCGCGATGGCGCAGCGCTCACGCTGCGTTCGGTCCAGTTCGCC1152                          AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GAGACCGATCTGTCCTTCGACTGCCTGACGCGGATGCCGGGCGCATCG1200                          GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ACCGTGGCCCAGCTCTACGACGCGACGCGCGGCTGGGGCCTGATCGAC1248                          ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  CATAACCTCGTCGCCGGGGATGTCGCGGGCTCGATCGGCCATCTGGTC1296                          HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     CGCGCCCGCGTTCCGTCCCGTCCGCGCGAAAACGGCTGGCTGCCGGTG1344                          ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     CCGGGCTGGTCCGGCGAGCATGAATGGCGGGGCTGGATTCCGCACGAG1392                          ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     GCGATGCCGCGCGTGATCGATCCGCCGGGCGGCATCATCGTCACGGCG1440                          AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AATAATCGCGTCGTGGCCGATGACCATCCCGATTATCTCTGCACCGAT1488                          AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  TGCCATCCGCCCTACCGCGCCGAGCGCATCATGAAGCGCCTGGTCGCC1536                          CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AATCCGGCTTTCGCCGTCGACGATGCCGCCGCGATCCATGCCGATACG1584                          AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     CTGTCGCCCCATGTCGGGTTGCTGCGCCGGAGGCTCGAGGCGCTTGGA1632                          LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     GCCCGCGACGACTCCGCGGCCGAAGGGCTGAGGCAGATGCTCGTCGCC1680                          AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TGGGACGGCCGCATGGATGCGGCTTCGGAGGTCGCGTCTGCCTACAAT1728                          TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  GCGTTCCGCAGGGCGCTGACGCGGCTGGTGACGGACCGCAGCGGGCTG1776                          AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GAGCAGGCGATATCGCATCCCTTCGCGGCTGTCGCGCCGGGCGTCTCA1824                          GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     CCGCAAGGCCAGGTCTGGTGGGCCGTGCCGACCCTGCTGCGCGACGAC1872                          ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     GATGCCGGAATGCTGAAGGGCTGGAGCTGGGACCAGGCCTTGTCTGAG1920                          AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     GCCCTCTCGGTCGCGTCGCAGAACCTGACCGGGCGAAGCTGGGGCGAA1968                          AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GAGCATCGGCCGCGCTTCACGCATCCGCTTGCCACGCAATTCCCGGCC2016                          GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TGGGCGGGGCTGCTGAATCCGGCTTCCCGTCCGATCGGTGGCGATGGC2064                          TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     GATACCGTGCTGGCGAACGGGCTCGTCCCGTCAGCCGGGCCGCAGGCG2112                          AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ACCTATGGTGCCCTGTCGCGCTACGTCTTCGATGTCGGCAATTGGGAC2160                          ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AATAGCCGCTGGGTCGTCTTCCACGGCGCCTCCGGGCATCCGGCCAGC2208                          AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  GCCCATTATGCCGATCAGAATGCGCCCTGGAGCGACTGTGCGATGGTG2256                          AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     CCGATGCTCTATAGCTGGGACAGGATCGCGGCAGAGGCCGTGACGTCG2304                          ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     CAGGAACTCGTCCCGGCCTGA2325                                                     GlnGluLeuValProAla*                                                           770                                                                           (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 774 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GluAlaAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GluAlaLysAspMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     LeuGlyLeuLeuMetGlyAlaValTrpPheLysLeuTrpArgMetLeu                              160165170175                                                                  AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  LeuAlaGlyAspProHisArgValPheGluIleProGlyMetTyrAla                              260265270                                                                     GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     ArgGluPheAspIleValGluThrArgHisGlyProValIleAlaGly                              355360365                                                                     AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     GlnGluLeuValProAla                                                            770                                                                           (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2325 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..2325                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 4..2322                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      ATGACTATGGCAGCTAATACGGATCGCGCGGTCTTGCAGGCGGCGCTG48                            MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       CCGCCGCTTTCCGGCAGCCTCCCCATTCCCGGATTGAGCGCGTCGGTC96                            ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        CGCGTCCGGCGCGATGCCTGGGGCATCCCGCATATCAAGGCCTCGGGC144                           ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GAGGCCGATGCCTATCGGGCGCTGGGCTTCGTCCATTCGCAGGACCGT192                           GluAlaAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        CTTTTCCAGATGGAGCTGACGCGTCGCAAGGCGCTGGGACGCGCGGCC240                           LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GAATGGCTGGGCGCCGAGGCCGCCGAGGCCGATATCCTCGTGCGCCGG288                           GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      CTCGGAATGGAAAAAGTCTGCCGGCGCGACTTCGAGGCCTTGGGCGTC336                           LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GAGGCGAAGGACATGCTGCGGGCTTATGTCGCCGGCGTGAACGCATTC384                           GluAlaLysAspMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     CTGGCTTCCGGTGCTCCCCTGCCTGTCGAATACGGATTGCTCGGAGCA432                           LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GAGCCGGAGCCCTGGGAGCCTTGGCACAGCATCGCGGTGATGCGCCGG480                           GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     CTGGGCCTGCTTATGGGTTCGGTGTGGTTCAAGCTCTGGCGGATGCTG528                           LeuGlyLeuLeuMetGlySerValTrpPheLysLeuTrpArgMetLeu                              160165170175                                                                  GCGCTGCCGGTGGTCGGAGCCGCGAATGCGCTGAAGCTGCGCTATGAC576                           AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     GATGGCGGCCGGGATTTGCTCTGCATCCCGCCGGGCGCCGAAGCCGAT624                           AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     CGGCTCGAGGCGGATCTCGCGACCCTGCGGCCCGCGGTCGATGCGCTG672                           ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     CTGAAGGCGATGGGCGGCGATGCCTCCGATGCTGCCGGCGGCGGCAGC720                           LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AACAACTGGGCGGTCGCTCCGGGCCGCACGGCGACCGGCAGGCCGATC768                           AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  CTCGCGGGCGATCCGCATCGCGTCTTCGAAATACCGGGCATCTATGCG816                           LeuAlaGlyAspProHisArgValPheGluIleProGlyIleTyrAla                              260265270                                                                     CAGCATCATCTGGCCTGCGACCGGTTCGACATGATCGGCCTGACCGTG864                           GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     CCGGGCGTGCCGGGCTTCCCGCACTTCGCGCATAACGGCAAGGTCGCC912                           ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TATTGCGTCACCCATGCCTTCATGGACATCCACGATCTCTATCTCGAG960                           TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     CAGTTCGCGGGGGAGGGCCGCACTGCGCGGTTCGGCAACGATTTCGAG1008                          GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  CCCGTCGCCTGGAGCCGGGACCGTATCGCGGTCCGGGGTGGCGCCGAT1056                          ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     CGCGAGTTCGATATAGTCAAGACGCGCCATGGCCCGGTTATCGCGGGC1104                          ArgGluPheAspIleValLysThrArgHisGlyProValIleAlaGly                              355360365                                                                     GATCCGCGCGATGGCGCAGCGCTCACGCTGCGTTCGGTCCAGTTCGCC1152                          AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GAGACCGATCTGTCCTTCGACTGCCTGACGCGGATGCCGGGCGCATCG1200                          GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ACCGTGGCCCAGCTCTACGACGCGACGCGCGGCTGGGGCCTGATCGAC1248                          ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  CATAACCTCGTCGCCGGGGATGTCGCGGGCTCGATCGGCCATCTGGTC1296                          HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     CGCGCCCGCGTTCCGTCCCGTCCGCGCGAAAACGGCTGGCTGCCGGTG1344                          ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     CCGGGCTGGTCCGGCGAGCATGAATGGCGGGGCTGGATTCCGCACGAG1392                          ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     GCGATGCCGCGCGTGATCGATCCGCCGGGCGGCATCATCGTCACGGCG1440                          AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AATAATCGCGTCGTGGCCGATGACCATCCCGATTATCTCTGCACCGAT1488                          AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  TGCCATCCGCCCTACCGCGCCGAGCGCATCATGAAGCGCCTGGTCGCC1536                          CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AATCCGGCTTTCGCCGTCGACGATGCCGCCGCGATCCATGCCGATACG1584                          AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     CTGTCGCCCCATGTCGGGTTGCTGCGCCGGAGGCTCGAGGCGCTTGGA1632                          LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     GCCCGCGACGACTCCGCGGCCGAAGGGCTGAGGCAGATGCTCGTCGCC1680                          AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TGGGACGGCCGCATGGATGCGGCTTCGGAGGTCGCGTCTGCCTACAAT1728                          TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  GCGTTCCGCAGGGCGCTGACGCGGCTGGTGACGGACCGCAGCGGGCTG1776                          AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GAGCAGGCGATATCGCATCCCTTCGCGGCTGTCGCGCCGGGCGTCTCA1824                          GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     CCGCAAGGCCAGGTCTGGTGGGCCGTGCCGACCCTGCTGCGCGACGAC1872                          ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     GATGCCGGAATGCTGAAGGGCTGGAGCTGGGACCAGGCCTTGTCTGAG1920                          AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     GCCCTCTCGGTCGCGTCGCAGAACCTGACCGGGCGAAGCTGGGGCGAA1968                          AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GAGCATCGGCCGCGCTTCACGCATCCGCTTGCCACGCAATTCCCGGCC2016                          GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TGGGCGGGGCTGCTGAATCCGGCTTCCCGTCCGATCGGTGGCGATGGC2064                          TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     GATACCGTGCTGGCGAACGGGCTCGTCCCGTCAGCCGGGCCGCAGGCG2112                          AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ACCTATGGTGCCCTGTCGCGCTACGTCTTCGATGTCGGCAATTGGGAC2160                          ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AATAGCCGCTGGGTCGTCTTCCACGGCGCCTCCGGGCATCCGGCCAGC2208                          AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  GCCCATTATGCCGATCAGAATGCGCCCTGGAGCGACTGTGCGATGGTG2256                          AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     CCGATGCTCTATAGCTGGGACAGGATCGCGGCAGAGGCCGTGACGTCG2304                          ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     CAGGAACTCGTCCCGGCCTGA2325                                                     GlnGluLeuValProAla*                                                           770                                                                           (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 774 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GluAlaAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GluAlaLysAspMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     LeuGlyLeuLeuMetGlySerValTrpPheLysLeuTrpArgMetLeu                              160165170175                                                                  AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  LeuAlaGlyAspProHisArgValPheGluIleProGlyIleTyrAla                              260265270                                                                     GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     ArgGluPheAspIleValLysThrArgHisGlyProValIleAlaGly                              355360365                                                                     AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     GlnGluLeuValProAla                                                            770                                                                           (2) INFORMATION FOR SEQ ID NO:49:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2325 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..2325                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 4..2322                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                      ATGACTATGGCAGCTAATACGGATCGCGCGGTCTTGCAGGCGGCGCTG48                            MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       CCGCCGCTTTCCGGCAGCCTCCCCATTCCCGGATTGAGCGCGTCGGTC96                            ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        CGCGTCCGGCGCGATGCCTGGGGCATCCCGCATATCAAGGCCTCGGGC144                           ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GAGGCCGATGCCTATCGGGCGCTGGGCTTCGTCCATTCGCAGGACCGT192                           GluAlaAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        CTTTTCCAGATGGAGCTGACGCGTCGCAAGGCGCTGGGACGCGCGGCC240                           LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GAATGGCTGGGCGCCGAGGCCGCCGAGGCCGATATCCTCGTGCGCCGG288                           GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      CTCGGAATGGAAAAAGTCTGCCGGCGCGACTTCGAGGCCTTGGGCGTC336                           LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GAGGCGAAGGACATGCTGCGGGCTTATGTCGCCGGCGTGAACGCATTC384                           GluAlaLysAspMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     CTGGCTTCCGGTGCTCCCCTGCCTGTCGAATACGGATTGCTCGGAGCA432                           LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GAGCCGGAGCCCTGGGAGCCTTGGCACAGCATCGCGGTGATGCGTCGA480                           GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     CTCGGCCTGCTTCTGGGATCCGTGTGGTTCAAGCTCTGGCGGGCGCTA528                           LeuGlyLeuLeuLeuGlySerValTrpPheLysLeuTrpArgAlaLeu                              160165170175                                                                  GCGCTGCCGGTGGTCGGAGCCGCGAATGCGCTGAAGCTGCGCTATGAC576                           AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     GATGGCGGCCGGGATTTGCTCTGCATCCCGCCGGGCGCCGAAGCCGAT624                           AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     CGGCTCGAGGCGGATCTCGCGACCCTGCGGCCCGCGGTCGATGCGCTG672                           ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     CTGAAGGCGATGGGCGGCGATGCCTCCGATGCTGCCGGCGGCGGCAGC720                           LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AACAACTGGGCGGTCGCTCCGGGCCGCACGGCGACCGGCAGGCCGATC768                           AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  CTCGCGGGCGATCCGCATCGCGTCTTCGAAATCCCTGGCTATTATGCG816                           LeuAlaGlyAspProHisArgValPheGluIleProGlyTyrTyrAla                              260265270                                                                     CAGCATCATCTGGCCTGCGACCGGTTCGACATGATCGGCCTGACCGTG864                           GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     CCGGGCGTGCCGGGCTTCCCGCACTTCGCGCATAACGGCAAGGTCGCC912                           ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TATTGCGTCACCCATGCCTTCATGGACATCCACGATCTCTATCTCGAG960                           TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     CAGTTCGCGGGGGAGGGCCGCACTGCGCGGTTCGGCAACGATTTCGAG1008                          GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  CCCGTCGCCTGGAGCCGGGACCGTATCGCGGTCCGGGGTGGCGCCGAT1056                          ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     CGCGAGTTCGATATCGTCGAGACGCGCCATGGCCCGGTTATCGCGGGC1104                          ArgGluPheAspIleValGluThrArgHisGlyProValIleAlaGly                              355360365                                                                     GATCCGCGCGATGGCGCAGCGCTCACGCTGCGTTCGGTCCAGTTCGCC1152                          AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GAGACCGATCTGTCCTTCGACTGCCTGACGCGGATGCCGGGCGCATCG1200                          GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ACCGTGGCCCAGCTCTACGACGCGACGCGCGGCTGGGGCCTGATCGAC1248                          ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  CATAACCTCGTCGCCGGGGATGTCGCGGGCTCGATCGGCCATCTGGTC1296                          HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     CGCGCCCGCGTTCCGTCCCGTCCGCGCGAAAACGGCTGGCTGCCGGTG1344                          ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     CCGGGCTGGTCCGGCGAGCATGAATGGCGGGGCTGGATTCCGCACGAG1392                          ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     GCGATGCCGCGCGTGATCGATCCGCCGGGCGGCATCATCGTCACGGCG1440                          AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AATAATCGCGTCGTGGCCGATGACCATCCCGATTATCTCTGCACCGAT1488                          AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  TGCCATCCGCCCTACCGCGCCGAGCGCATCATGAAGCGCCTGGTCGCC1536                          CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AATCCGGCTTTCGCCGTCGACGATGCCGCCGCGATCCATGCCGATACG1584                          AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     CTGTCGCCCCATGTCGGGTTGCTGCGCCGGAGGCTCGAGGCGCTTGGA1632                          LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     GCCCGCGACGACTCCGCGGCCGAAGGGCTGAGGCAGATGCTCGTCGCC1680                          AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TGGGACGGCCGCATGGATGCGGCTTCGGAGGTCGCGTCTGCCTACAAT1728                          TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  GCGTTCCGCAGGGCGCTGACGCGGCTGGTGACGGACCGCAGCGGGCTG1776                          AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GAGCAGGCGATATCGCATCCCTTCGCGGCTGTCGCGCCGGGCGTCTCA1824                          GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     CCGCAAGGCCAGGTCTGGTGGGCCGTGCCGACCCTGCTGCGCGACGAC1872                          ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     GATGCCGGAATGCTGAAGGGCTGGAGCTGGGACCAGGCCTTGTCTGAG1920                          AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     GCCCTCTCGGTCGCGTCGCAGAACCTGACCGGGCGAAGCTGGGGCGAA1968                          AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GAGCATCGGCCGCGCTTCACGCATCCGCTTGCCACGCAATTCCCGGCC2016                          GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TGGGCGGGGCTGCTGAATCCGGCTTCCCGTCCGATCGGTGGCGATGGC2064                          TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     GATACCGTGCTGGCGAACGGGCTCGTCCCGTCAGCCGGGCCGCAGGCG2112                          AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ACCTATGGTGCCCTGTCGCGCTACGTCTTCGATGTCGGCAATTGGGAC2160                          ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AATAGCCGCTGGGTCGTCTTCCACGGCGCCTCCGGGCATCCGGCCAGC2208                          AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  GCCCATTATGCCGATCAGAATGCGCCCTGGAGCGACTGTGCGATGGTG2256                          AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     CCGATGCTCTATAGCTGGGACAGGATCGCGGCAGAGGCCGTGACGTCG2304                          ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     CAGGAACTCGTCCCGGCCTGA2325                                                     GlnGluLeuValProAla*                                                           770                                                                           (2) INFORMATION FOR SEQ ID NO:50:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 774 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                      MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GluAlaAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GluAlaLysAspMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     LeuGlyLeuLeuLeuGlySerValTrpPheLysLeuTrpArgAlaLeu                              160165170175                                                                  AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  LeuAlaGlyAspProHisArgValPheGluIleProGlyTyrTyrAla                              260265270                                                                     GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     ArgGluPheAspIleValGluThrArgHisGlyProValIleAlaGly                              355360365                                                                     AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     GlnGluLeuValProAla                                                            770                                                                           (2) INFORMATION FOR SEQ ID NO:51:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2325 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..2325                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: mat.sub.-- peptide                                              (B) LOCATION: 4..2322                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                      ATGACTATGGCAGCTAATACGGATCGCGCGGTCTTGCAGGCGGCGCTG48                            MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       CCGCCGCTTTCCGGCAGCCTCCCCATTCCCGGATTGAGCGCGTCGGTC96                            ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        CGCGTCCGGCGCGATGCCTGGGGCATCCCGCATATCAAGGCCTCGGGC144                           ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GAGCTCGATGCCTATCGGGCGCTGGGCTTCGTCCATTCGCAGGACCGT192                           GluLeuAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        CTTTTCCAGATGGAGCTGACGCGTCGCAAGGCGCTGGGACGCGCGGCC240                           LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GAATGGCTGGGCGCCGAGGCCGCCGAGGCCGATATCCTCGTGCGCCGG288                           GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      CTCGGAATGGAAAAAGTCTGCCGGCGCGACTTCGAGGCCTTGGGCGTC336                           LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GAGGCGAAGGAGATGCTGCGGGCTTATGTCGCCGGCGTGAACGCATTC384                           GluAlaLysGluMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     CTGGCTTCCGGTGCTCCCCTGCCTGTCGAATACGGATTGCTCGGAGCA432                           LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GAGCCGGAGCCCTGGGAGCCTTGGCACAGCATCGCGGTGATGCGCCGG480                           GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     CTGGGCCTGCTTATGGGTTCGGTGTGGTTCAAGCTCTGGCGGATGCTG528                           LeuGlyLeuLeuMetGlySerValTrpPheLysLeuTrpArgMetLeu                              160165170175                                                                  GCGCTGCCGGTGGTCGGAGCCGCGAATGCGCTGAAGCTGCGCTATGAC576                           AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     GATGGCGGCCGGGATTTGCTCTGCATCCCGCCGGGCGCCGAAGCCGAT624                           AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     CGGCTCGAGGCGGATCTCGCGACCCTGCGGCCCGCGGTCGATGCGCTG672                           ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     CTGAAGGCGATGGGCGGCGATGCCTCCGATGCTGCCGGCGGCGGCAGC720                           LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AACAACTGGGCGGTCGCTCCGGGCCGCACGGCGACCGGCAGGCCGATC768                           AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  CTCGCGGGCGATCCGCATCGCGTCTTCGAAATCCCGGGCATGTATGCG816                           LeuAlaGlyAspProHisArgValPheGluIleProGlyMetTyrAla                              260265270                                                                     CAGCATCATCTGGCCTGCGACCGGTTCGACATGATCGGCCTGACCGTG864                           GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     CCGGGCGTGCCGGGCTTCCCGCACTTCGCGCATAACGGCAAGGTCGCC912                           ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TATTGCGTCACCCATGCCTTCATGGACATCCACGATCTCTATCTCGAG960                           TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     CAGTTCGCGGGGGAGGGCCGCACTGCGCGGTTCGGCAACGATTTCGAG1008                          GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  CCCGTCGCCTGGAGCCGGGACCGTATCGCGGTCCGGGGTGGCGCCGAT1056                          ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     CGCGAGTTCGATATCGTCGAGACGCGCCATGGCCCGGTTATCGCGGGC1104                          ArgGluPheAspIleValGluThrArgHisGlyProValIleAlaGly                              355360365                                                                     GATCCGCGCGATGGCGCAGCGCTCACGCTGCGTTCGGTCCAGTTCGCC1152                          AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GAGACCGATCTGTCCTTCGACTGCCTGACGCGGATGCCGGGCGCATCG1200                          GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ACCGTGGCCCAGCTCTACGACGCGACGCGCGGCTGGGGCCTGATCGAC1248                          ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  CATAACCTCGTCGCCGGGGATGTCGCGGGCTCGATCGGCCATCTGGTC1296                          HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     CGCGCCCGCGTTCCGTCCCGTCCGCGCGAAAACGGCTGGCTGCCGGTG1344                          ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     CCGGGCTGGTCCGGCGAGCATGAATGGCGGGGCTGGATTCCGCACGAG1392                          ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     GCGATGCCGCGCGTGATCGATCCGCCGGGCGGCATCATCGTCACGGCG1440                          AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AATAATCGCGTCGTGGCCGATGACCATCCCGATTATCTCTGCACCGAT1488                          AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  TGCCATCCGCCCTACCGCGCCGAGCGCATCATGAAGCGCCTGGTCGCC1536                          CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AATCCGGCTTTCGCCGTCGACGATGCCGCCGCGATCCATGCCGATACG1584                          AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     CTGTCGCCCCATGTCGGGTTGCTGCGCCGGAGGCTCGAGGCGCTTGGA1632                          LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     GCCCGCGACGACTCCGCGGCCGAAGGGCTGAGGCAGATGCTCGTCGCC1680                          AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TGGGACGGCCGCATGGATGCGGCTTCGGAGGTCGCGTCTGCCTACAAT1728                          TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  GCGTTCCGCAGGGCGCTGACGCGGCTGGTGACGGACCGCAGCGGGCTG1776                          AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GAGCAGGCGATATCGCATCCCTTCGCGGCTGTCGCGCCGGGCGTCTCA1824                          GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     CCGCAAGGCCAGGTCTGGTGGGCCGTGCCGACCCTGCTGCGCGACGAC1872                          ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     GATGCCGGAATGCTGAAGGGCTGGAGCTGGGACCAGGCCTTGTCTGAG1920                          AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     GCCCTCTCGGTCGCGTCGCAGAACCTGACCGGGCGAAGCTGGGGCGAA1968                          AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GAGCATCGGCCGCGCTTCACGCATCCGCTTGCCACGCAATTCCCGGCC2016                          GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TGGGCGGGGCTGCTGAATCCGGCTTCCCGTCCGATCGGTGGCGATGGC2064                          TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     GATACCGTGCTGGCGAACGGGCTCGTCCCGTCAGCCGGGCCGCAGGCG2112                          AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ACCTATGGTGCCCTGTCGCGCTACGTCTTCGATGTCGGCAATTGGGAC2160                          ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AATAGCCGCTGGGTCGTCTTCCACGGCGCCTCCGGGCATCCGGCCAGC2208                          AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  GCCCATTATGCCGATCAGAATGCGCCCTGGAGCGACTGTGCGATGGTG2256                          AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     CCGATGCTCTATAGCTGGGACAGGATCGCGGCAGAGGCCGTGACGTCG2304                          ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     CAGGAACTCGTCCCGGCCTGA2325                                                     GlnGluLeuValProAla*                                                           770                                                                           (2) INFORMATION FOR SEQ ID NO:52:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 774 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                      MetThrMetAlaAlaAsnThrAspArgAlaValLeuGlnAlaAlaLeu                              1151015                                                                       ProProLeuSerGlySerLeuProIleProGlyLeuSerAlaSerVal                              202530                                                                        ArgValArgArgAspAlaTrpGlyIleProHisIleLysAlaSerGly                              354045                                                                        GluLeuAspAlaTyrArgAlaLeuGlyPheValHisSerGlnAspArg                              505560                                                                        LeuPheGlnMetGluLeuThrArgArgLysAlaLeuGlyArgAlaAla                              657075                                                                        GluTrpLeuGlyAlaGluAlaAlaGluAlaAspIleLeuValArgArg                              80859095                                                                      LeuGlyMetGluLysValCysArgArgAspPheGluAlaLeuGlyVal                              100105110                                                                     GluAlaLysGluMetLeuArgAlaTyrValAlaGlyValAsnAlaPhe                              115120125                                                                     LeuAlaSerGlyAlaProLeuProValGluTyrGlyLeuLeuGlyAla                              130135140                                                                     GluProGluProTrpGluProTrpHisSerIleAlaValMetArgArg                              145150155                                                                     LeuGlyLeuLeuMetGlySerValTrpPheLysLeuTrpArgMetLeu                              160165170175                                                                  AlaLeuProValValGlyAlaAlaAsnAlaLeuLysLeuArgTyrAsp                              180185190                                                                     AspGlyGlyArgAspLeuLeuCysIleProProGlyAlaGluAlaAsp                              195200205                                                                     ArgLeuGluAlaAspLeuAlaThrLeuArgProAlaValAspAlaLeu                              210215220                                                                     LeuLysAlaMetGlyGlyAspAlaSerAspAlaAlaGlyGlyGlySer                              225230235                                                                     AsnAsnTrpAlaValAlaProGlyArgThrAlaThrGlyArgProIle                              240245250255                                                                  LeuAlaGlyAspProHisArgValPheGluIleProGlyMetTyrAla                              260265270                                                                     GlnHisHisLeuAlaCysAspArgPheAspMetIleGlyLeuThrVal                              275280285                                                                     ProGlyValProGlyPheProHisPheAlaHisAsnGlyLysValAla                              290295300                                                                     TyrCysValThrHisAlaPheMetAspIleHisAspLeuTyrLeuGlu                              305310315                                                                     GlnPheAlaGlyGluGlyArgThrAlaArgPheGlyAsnAspPheGlu                              320325330335                                                                  ProValAlaTrpSerArgAspArgIleAlaValArgGlyGlyAlaAsp                              340345350                                                                     ArgGluPheAspIleValGluThrArgHisGlyProValIleAlaGly                              355360365                                                                     AspProArgAspGlyAlaAlaLeuThrLeuArgSerValGlnPheAla                              370375380                                                                     GluThrAspLeuSerPheAspCysLeuThrArgMetProGlyAlaSer                              385390395                                                                     ThrValAlaGlnLeuTyrAspAlaThrArgGlyTrpGlyLeuIleAsp                              400405410415                                                                  HisAsnLeuValAlaGlyAspValAlaGlySerIleGlyHisLeuVal                              420425430                                                                     ArgAlaArgValProSerArgProArgGluAsnGlyTrpLeuProVal                              435440445                                                                     ProGlyTrpSerGlyGluHisGluTrpArgGlyTrpIleProHisGlu                              450455460                                                                     AlaMetProArgValIleAspProProGlyGlyIleIleValThrAla                              465470475                                                                     AsnAsnArgValValAlaAspAspHisProAspTyrLeuCysThrAsp                              480485490495                                                                  CysHisProProTyrArgAlaGluArgIleMetLysArgLeuValAla                              500505510                                                                     AsnProAlaPheAlaValAspAspAlaAlaAlaIleHisAlaAspThr                              515520525                                                                     LeuSerProHisValGlyLeuLeuArgArgArgLeuGluAlaLeuGly                              530535540                                                                     AlaArgAspAspSerAlaAlaGluGlyLeuArgGlnMetLeuValAla                              545550555                                                                     TrpAspGlyArgMetAspAlaAlaSerGluValAlaSerAlaTyrAsn                              560565570575                                                                  AlaPheArgArgAlaLeuThrArgLeuValThrAspArgSerGlyLeu                              580585590                                                                     GluGlnAlaIleSerHisProPheAlaAlaValAlaProGlyValSer                              595600605                                                                     ProGlnGlyGlnValTrpTrpAlaValProThrLeuLeuArgAspAsp                              610615620                                                                     AspAlaGlyMetLeuLysGlyTrpSerTrpAspGlnAlaLeuSerGlu                              625630635                                                                     AlaLeuSerValAlaSerGlnAsnLeuThrGlyArgSerTrpGlyGlu                              640645650655                                                                  GluHisArgProArgPheThrHisProLeuAlaThrGlnPheProAla                              660665670                                                                     TrpAlaGlyLeuLeuAsnProAlaSerArgProIleGlyGlyAspGly                              675680685                                                                     AspThrValLeuAlaAsnGlyLeuValProSerAlaGlyProGlnAla                              690695700                                                                     ThrTyrGlyAlaLeuSerArgTyrValPheAspValGlyAsnTrpAsp                              705710715                                                                     AsnSerArgTrpValValPheHisGlyAlaSerGlyHisProAlaSer                              720725730735                                                                  AlaHisTyrAlaAspGlnAsnAlaProTrpSerAspCysAlaMetVal                              740745750                                                                     ProMetLeuTyrSerTrpAspArgIleAlaAlaGluAlaValThrSer                              755760765                                                                     GlnGluLeuValProAla                                                            770                                                                           (2) INFORMATION FOR SEQ ID NO:53:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                      CCGGGTGTGTACACCAAGGTTACCAACTACCTAGACTGGATTCGTGACAACATGCGACCG60                TGA63                                                                         (2) INFORMATION FOR SEQ ID NO:54:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                      AGCTTCACGGTCGCATGTTGTCACGAATCCAGTCTAGGTAGTTGGTAACCTTGGTGTACA60                CAC63                                                                         (2) INFORMATION FOR SEQ ID NO:55:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                      AGCTTGTCCTCGAGATCAAATAAAGGCTCCTTTTGGAGCCTTTTTTTTTTG51                         (2) INFORMATION FOR SEQ ID NO:56:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 51 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                      TCGACAAAAAAAAAAGGCTCCAAAAGGAGCCTTTAATTGATCTCGAGGACA51                         (2) INFORMATION FOR SEQ ID NO:57:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                      AATTCGGATCCAAGCTTA18                                                          (2) INFORMATION FOR SEQ ID NO:58:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                      CGCGTAAGCTTGGATCCG18                                                          (2) INFORMATION FOR SEQ ID NO:59:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                      XaaThrMetAlaAlaAsnThr                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:60:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                      CGATAAAATGACTATGGCGGCCAACACC28                                                (2) INFORMATION FOR SEQ ID NO:61:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                      GATCGGTGTTGGCCGCCATAGTCATTTTAT30                                              (2) INFORMATION FOR SEQ ID NO:62:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                      CGATAAAATGACTATGGCAGCTAATACG28                                                (2) INFORMATION FOR SEQ ID NO:63:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                      GATCCGTATTAGCTGCCATAGTCATTTTAT30                                              (2) INFORMATION FOR SEQ ID NO:64:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                      GACCGGCAGCGCTAGCGCCCGCCAGAGCTTGA32                                            __________________________________________________________________________

We claim:
 1. A mutant CC acylase wherein at least one amino acid at theAla⁴⁹, Met¹⁶⁴, Ser¹⁶⁶, Met¹⁷⁴, Glu³⁵⁸, Met⁴⁶⁵, Met⁵⁰⁶, or Met⁷⁵⁰position of the amino acid sequence of the native CC acylase is replacedby a different amino acid.
 2. A mutant Cephalosporin C acylase of claim1, which is represented by the following formula in its precursor formbefore processing into α-subunit and β-subunitthereof:A1-48-X1-A50-163-X2-Gly-X3-A167-173-X4-A175-357-X5-A359-464-X6-A466-505-X7-A507-749-X8-A751-773whereinA1-48 is the same amino acid sequence as that from Thr¹ to Glu⁴⁸ ofnative CC acylase, A50-163 is the same amino acid sequence as that fromAsp⁵⁰ to Leu¹⁶³ of native CC acylase, A167-173 is the same amino acidsequence as that from Val¹⁶⁷ to Arg¹⁷³ of native CC acylase, A175-357 isthe same amino acid sequence as that from Leu¹⁷⁵ to Val³⁵⁷ of native CCacylase, A359-464 is the same amino acid sequence as that from Thr³⁵⁹ toAla⁴⁶⁴ of native CC acylase, A466-505 is the same amino acid sequence asthat from Pro⁴⁶⁶ to Ile⁵⁰⁵ of native CC acylase, A507-749 is the sameamino acid sequence as that from Lys⁵⁰⁷ to Ala⁷⁴⁹ of native CC acylase,A751-773 is the same amino acid sequence as that from Val⁷⁵¹ to Ala⁷⁷³of native CC acylase, X1 is Ala or a different amino acid, X2, X4, X6,X7 and X8 are each Met or a different amino acid, X3 is Ser or adifferent amino acid and X5 is Glu or a different amino acid,providingthat Met²⁶⁹ and/or Cys³⁰⁵ may be replaced by (a) different amino acid(s)in the above formula, and when X1 is Ala, X2, X4, X6, X7 and X8 are eachMet, X3 is Ser and X5 is an amino acid other than Glu.
 3. A mutantCephalosporin C acylase of claim 2, in which X1 is leucine, X3 isalanine, and Met²⁶⁹ is replaced by tyrosine.
 4. An isolated DNA whichencodes the Cephalosporin C acylase of claim
 1. 5. An expression vectorwhich comprises the DNA of claim
 4. 6. A host cell transformed by theexpression vector of claim
 5. 7. A process for producing a mutantCephalosporin C acylase, which comprises culturing a host celltransformed by an expression vector which comprises DNA which encodesthe Cephalosporin C acylase of claim 1 in an aqueous nutrient medium andrecovering the mutant Cephalosporin C acylase from the cultured broth.8. A process for preparing a compound of the formula (I): ##STR3##wherein R¹ is acetoxy, hydroxy, or hydrogen, or its salt,which comprisescontacting a compound of the formula (II): ##STR4## wherein R¹ is thesame as defined above and R² is carboxylic acyl, or its saltwith thecultured broth of the transformant of claim 6 or its processed material.